Formulations and methods of use for alpha connexin C-terminal (ACT) peptides

ABSTRACT

This invention relates to a topical gel drug product preparation containing a composition comprising an isolated polypeptide having a carboxy-terminal amino acid sequence of an alpha connexin (ACT peptide), peptide stabilizers, excipients, buffering agents, and the like. A formulation and preparation steps are disclosed for the manufacturing of a stable, elegant, and pourable topical gel. The resulting formulation possesses long term stability suitable for aesthetic as well as therapeutic applications including the prevention of scaring and accelerated healing of wounds. Methods for treatment of chronic wounds, including chronic ulcers, are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT Application NO. PCT/US2013/028727, filed Mar. 1, 2013, which claims priority to U.S. Provisional Patent Application No. 61/605,528, filed Mar. 1, 2012, which is incorporated herein by reference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: FIRS_(—)006_(—)01US_SeqList_ST25.txt, date recorded: Jun. 17, 2013, file size 32 kilobytes).

TECHNICAL FIELD

The present invention relates to compositions for wound treatment comprising alpha connexin polypeptides and stabilizing agents. Topical formulations containing alpha connexin polypeptides and hydroxyethylcellulose are particularly stable. These compositions may be used to treat a variety of wounds including acute surgical wounds and chronic ulcers. Methods for treatment of chronic wounds are also provided.

BACKGROUND

While human tissues damaged by mechanical wounding, disease processes and other causes are capable of healing, complex tissue structure and function is rarely, if ever wholly restored. Instead, recovery of nearly all tissues from injury in humans and other higher vertebrates is dominated by the formation of scar tissue. The most familiar example of this is the discolored and fibrotic scars that linger following the healing of a skin cut or graze. Less well appreciated is that formation of glial scar tissue following injury to the brain or spinal chord is one of the main obstacles to restoration of neural function following damage to the central nervous system (Silver and Miller J H, 2004). There is currently no means of treating or preventing such scarring and promoting the regeneration of complex tissue structure and function following injury.

Once such treatments are discovered, there is also a need to provide formulations that stabilize the active pharmaceutical ingredients (API) which they contain. While there are a myriad of potential stabilizing agents, not all of them work equally well in stabilizing specific APIs.

Venous ulcers are chronic wounds associated with long-standing venous hypertension of the lower extremity. The number of individuals affected by these ulcers in the United States is over 600,000. These indolent wounds are a major cause of morbidity and are a major financial burden. Diabetes affects an estimated 25.8 million people (8% of the US population) according to the Centers for Disease Control and Prevention. People with diabetes have 12-25% chance of developing foot ulcer in their lifetime. Diabetic Foot Ulcers (DFUs) refer to wounds below the ankle of a diabetic subject which develops due to neuropathy (sensory, motor, or autonomic deficits), ischemia or both. Foot ulcers are a substantial cause for morbidity and can severely impair quality of life, engender high treatment costs, and serve as the most important risk factor for lower-extremity amputation.

No single available product or procedure is adequate for the treatment of all subjects with chronic ulcers. Treatment selection is determined by patient tolerance, patient medical status, cost, availability, and physician preference. Compression wraps are the Standard of Care (SoC) and are needed to assist venous return and to address the underlying pathophysiology of venous disease and venous ulceration. Compression bandages and stockings heal more ulcers compared with no compression, but it has not been ascertained if intermittent pneumatic compression is beneficial compared with compression bandages or stockings. Occlusive (hydrocolloid) dressings are no more effective than simple low-adherent dressings in people treated with compression.

Despite various advances in wound treatment, the need exists for additional effective VLU and DFU treatments that combine advanced wound care therapeutics with SoC compression therapy for faster healing of ulcers. Rapid healing would result in fewer hospitalizations, decreased infection, less use of antibiotics, improved mobility, less pain, and fewer surgical procedures for subjects with VLUs or DFUs.

SUMMARY OF THE INVENTION

The present invention is directed in part to a topical gel drug product preparation containing a composition comprising an isolated alpha connexin polypeptide. The topical formulation may further comprise hydroxyethylcellulose gel, which stabilizes the alpha connexin polypeptide during storage. In certain embodiments the alpha connexin polypeptide comprises a carboxy terminal amino acid sequence of an alpha connexin (hereinafter “ACT”). The alpha connexin polypeptides of the present invention may comprise or consist of the carboxy-terminal most 4 to 30 contiguous amino acids of an alpha connexin protein or conservative variant thereof, wherein said at least one alpha connexin polypeptide is linked at its amino terminus to a cellular internalization transporter.

It is an object of the present invention to prepare a formulation of a stable, elegant, and pourable topical gel carrier that contains ACT for aesthetic as well as therapeutic applications including the prevention of scaring and accelerated healing of wounds of a subject. The drug product may be administered to treat acute surgical wounds to reduce scarring or be applied chronically to difficult-to-heal wounds including, but not limited to, venous leg ulcers, diabetic foot ulcers, pressure ulcers, and the like. The drug product possesses physicochemical, biochemical, and rheological properties that enable its ability to provide a therapeutic and effective amount of ACT peptide when applied to all type of wounds.

In one aspect of the invention, one or more stabilizer is selected among many, and prepared with ACT peptide to stabilize the isolated polypeptide. Preferably the stabilizers are non-irritating, non-staining, and non-immunogenic. The stabilizers enable the long term (i.e., for 3 months, for 6 months, for 9 months, for 12 months, for 18 months, or for 24 months) storage of the drug product under a variety of temperature conditions (i.e., at about 5° C., at about 10° C., at about 15° C., at about 20° C., at about 25° C., at about 30° C., at about 35° C., or at about 40° C.) and under a range of relative humidities (i.e., at 0% relative humidity, at 10% relative humidity, at 20% relative humidity, at 30% relative humidity, at 40% relative humidity, at 50% relative humidity, at 60% relative humidity, at 70% relative humidity, at 80% relative humidity, at 90% relative humidity, or at 100% relative humidity).

In another aspect of the invention, one or more excipients are selected from the group consisting of water-soluble porogens, polymers, ionic compounds, non-ionic compounds, and solvents. In a further embodiment, excipients act as one or more of carriers, stabilizers, and gelling agents. In one embodiment, at least one excipient is prepared with ACT. Preferably the excipients are non-irritating, non-staining, and non-immunogenic. In one embodiment the formulation comprises at least one polymer. In a further embodiment, the formulation comprises a derivative of cellulose. In a yet further embodiment, the derivative of cellulose is hydroxyethylcellulose.

In yet another aspect of the invention, an optimal pH range for the topical gel is about pH 5 to about pH 7, and is maintained by the addition of one or more buffering agents. Preferably the buffering agents are non-irritating, non-staining, and non-immunogenic.

In yet another aspect of the invention, at least one excipient is a gelling agent. In one embodiment, a gelling agent is a carrier of ACT. In some embodiments, the gelling agent also acts as a stabilizer. In some embodiments, the gelling agent may be combined with at least one of the following: one or more buffering agent, and one or more additional excipient. In one embodiment, the gelling agent is hydroxyethylcellulose.

In some embodiments, the present invention includes a method of wound treatment comprising administering to a subject in need thereof a topical formulation comprising at least one alpha connexin polypeptide and hydroxyethylcellulose gel. In an exemplary embodiment, the steps are disclosed for manufacturing of the drug product.

The present invention also includes methods of treating wounds and ulcers in a patient in need thereof, particularly wounds and ulcers that are chronic in nature. The present inventors have found that the peptides and formulations of the present invention provide faster healing of ulcers when combined with compression therapy than is achieved by compression therapy alone. Accordingly, the present invention includes methods of treating a chronic wound in a subject, comprising administering to the subject a topical formulation comprising at least one alpha connexin polypeptide in addition to standard of care compression therapy, wherein the chronic wound is healed at a faster rate and/or increased frequency than achieved with standard of care compression therapy alone.

In one aspect a method of treating a chronic wound in a subject comprising administering to the subject a topical formulation comprising at least one alpha connexin polypeptide is provided. In a further embodiment, the formulation is administered daily or weekly or a combination thereof. In another embodiment, the formulation is administered according to any dosing regiment that is effective in the treatment of the chronic wound. In a further embodiment, the formulation is administered in a dosing regimen at day 0, day 3, week 1, week 2, week 3, week 4, week 5, week 6, week 7, week 8, week 9, week 10, week 11, and week 12.

In one embodiment a method of treating a chronic wound in a subject comprising administering to the subject a topical formulation comprising at least one alpha connexin polypeptide and hydroxyethylcellulose is provided. In a further embodiment, the hydroxyethylcellulose is present at a concentration of about 1.25% (w/w). In one embodiment, the at least one isolated polypeptide comprises the carboxy terminal-most 4 to 30 contiguous amino acids of an alpha Connexin, or a conservative variant thereof. In another embodiment, the alpha connexin polypeptide is connexin 37, connexin 40, connexin 43, or connexin 45. In one embodiment, the alpha connexin is linked at its amino terminus to a cellular internalization transporter such as, for example an Antennapedia sequence, TAT, HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II, Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynB1, Pep-7, FIN-1, BGSC (Bis-Guanidinium-Spermidine-Cholesterol, and BGTC (BisGuanidinium-Tren-Cholesterol). In one embodiment, the cellular internalization transporter is an antennapedia sequence. In a further embodiment, the formulation comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 9.

In one embodiment, the formulation for use in treatment of a chronic wound comprises at least one alpha connexin polypeptide and hydroxyethylcellulose gel. In a further embodiment, the hydroxycellulose gel is present at a concentration of about 2%, about 1.75%, about 1.5%, about 1.25%, about 1.0%, or about 0.75%. In one embodiment, the hydroxycellulose gel is present at a concentration of about 1.25% (w/w).

In one aspect, the chronic wound is an ulcer. In a further embodiment, the chronic wound is a lower extremity ulcer. In another embodiment, the chronic wound is selected from the group consisting of venous leg ulcers, diabetic foot ulcers, and pressure ulcers. In one embodiment, a method for treating a chronic wound in a subject is provided, wherein the method comprises administering to the subject a topical formulation of at least one alpha connexin polypeptide, wherein the formulation is administered in a dosing regimen at day 0, day 3, week 1, week 2, week 3, week 4, week 5, week 6, week 7, week 8, week 9, week 10, week 11, and week 12, wherein the symptoms of the chronic wound are reduced. In a further embodiment, the formulation does not induce excessive levels of side effects. In another embodiment, the chronic wound is improved in the absence of clinically significant abnormalities. In another embodiment, the method reduces the time to 100% wound closure, as compared to the time to 100% wound closure when the standard of care treatment is used. In other embodiments, the method reduces the time to 100% wound closure when administered in conjunction with standard of care, as compared to treatment with either standard of care alone.

Standard of care treatments are known to those of skill in the art and include, for example, cleaning the wound, applying dressing to the wound, and applying a pressure bandage to the wound. In one embodiment, the subject treated with the methods and formulations provided herein has a higher percent wound closure as compared to a subject receiving standard of care therapy, at 12 weeks, 11 weeks, 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, or 2 weeks. In one embodiment, the percent wound closure at 12 weeks is higher in subjects treated with the methods and formulations disclosed herein, as compared to the percent wound closure at 12 weeks in subjects receiving standard of care treatment. In one embodiment, the method reduces the time to 50% wound closure, as compared to the time to 50% wound closure when the standard of care treatment is used. In another embodiment, the percent wound closure at 4 weeks is higher in subjects treated with the methods and formulations disclosed herein, as compared to the percent wound closure at 4 weeks in subjects treated with standard of care treatment.

In another embodiment, the method results in a reduction in pain levels in the subject. In a further embodiment, the pain level is determined through patient self-assessment. In one embodiment, the method increases the average percent of wound closure at 12 weeks, 11 weeks, 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, or 2 weeks as compared to standard of care treatment. In one embodiment, the method increases the average percent of wound closure at 12 weeks. In one embodiment, the method decreases the wound area as compared to the wound area in subjects that are treated with standard of care therapy. In one embodiment, the method does not induce the production of anti-alpha connexin polypeptide antibodies in the subject.

In another embodiment, the method increases the incidence or frequency of 100% complete wound closure compared to standard of care treatments for wound healing. In another embodiment, the method is used to treat an ulcer lacking sufficient wound size reduction within one, four, 12, 24, 36, or more weeks of standard of care. In one embodiment, the method is used to treat an ulcer with less than 50% wound closure within one four, 12, 24, 36, or more weeks of standard of care. In one embodiment, the method is used to treat an ulcer lacking sufficient wound size reduction within one, four, 12, 24, 36, or more weeks of standard of care. In one embodiment, the method is used to treat an ulcer lacking sufficient wound size reduction within one, four, 12, 24, 36, or more weeks of standard of care. In one embodiment, the method is used to treat a patient with an ulcer possessing wound area and duration characteristics of a chronic wound. In one embodiment, the method is used to treat a patient with an ulcer possessing a wound with a non-healing wound trajectory. In one embodiment, the method is used to treat an ulcer possessing wound area and time duration characteristics of a chronic wound.

DESCRIPTION OF DRAWINGS

For a fuller understanding of the nature and advantages of the present invention, reference should be had to the following detailed description taken in connection with the accompanying drawings, in which:

FIG. 1 illustrates the manufacturing process flow chart.

FIG. 2 shows the subject self assessment of pain in the intent-to-treat population in the Diabetic Foot Ulcer (DFU) study.

FIG. 3 shows the subject self assessment of pain in the per-protocol (PP) population in the DFU study.

FIG. 4 shows the time to 100% wound closure in the intent-to-treat (ITT) population in the DFU study.

FIG. 5 shows the time to 100% wound closure in the PP population in the DFU study.

FIG. 6 shows the time to 50% wound closure in the ITT population in the DFU study.

FIG. 7 shows the time to 50% wound closure in the PP population in the DFU study.

FIG. 8 shows the average wound closure at each visit in the ITT population in the DFU study.

FIG. 9 shows the average wound closure at each visit in the PP population in the DFU study.

FIG. 10 shows the Longitudinal Response Profile of the Mean Percent Reduction of Wound Area (mm²) from Baseline to Week 12 by Treatment Group—ITT Population (N=92)

FIG. 11 Longitudinal Response Profile of the Mean Percentage Reduction of Wound Area from Baseline to Week 12 by Treatment Group—mITT Population (N=77)

FIG. 12 shows the Longitudinal Response Profile of the Mean Percentage Reduction of Wound Area (mm²) from Baseline to Week 12 by Treatment Group—PP Population (N=68)

FIG. 13 shows the Kaplan-Meier Plot of Time to First 100% Wound Closure ITT Population (N=92)

FIG. 14 shows the Kaplan-Meier Plot of Time to First 100% Wound Closure ITT Population (N=92)

FIG. 15 shows Kaplan-Meier Plot of Time to First 100% Wound Closure PP Population (N=68)

FIG. 16 shows the Kaplan-Meier Plot of Time to First 50% Wound Closure ITT Population (N=92)

FIG. 17 shows the Kaplan-Meier Plot of Time to 50% Wound Closure—mITT Population (N=77)

FIG. 18 shows the Kaplan-Meier Plot of Time to First 50% Wound Closure PP Population (N=68)

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and it is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

It must also be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to an “element” is a reference to one or more elements and equivalents thereof known to those skilled in the art, and so forth.

The term “topical” refers to administration of inventive drug product at, or immediately beneath, the point of application. As used herein “topical application” refers to application onto one or more surfaces(s) including keratinous tissue, i.e., “topically applying.” Topical application or “topically applying” may involve direct application to the area of the desired substrate. The topical preparation and/or composition may be applied by pouring, dropping, or spraying, if a liquid; rubbing on, if an ointment, lotion, cream, gel, or the like; dusting, if a powder; spraying, if a liquid or aerosol composition; or by any other appropriate means.

As used herein, the phrase “gelling agents” refers to agents that make a topical preparation denser or more viscous in consistency. Gelling agents may be water-based or oil-based. Gelling agents may also be referred to as “thickening agents.” “Excipients,” as used herein, are inactive ingredients that serve as carriers of the active pharmaceutical ingredient (API). Excipients may also serve as gelling or thickening agents, or stabilizers.

A “subject” or a “mammal” includes a human or a non-human mammal. Non-human mammals include, but not limited to, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In one embodiment, the subject or mammal is human.

“Administering” when used in conjunction with a therapeutic means to administer a therapeutic directly into or onto a target tissue or to administer a therapeutic to a patient whereby the therapeutic positively impacts the tissue to which it is targeted. Thus, as used herein, the term “administering”, when used in conjunction with the drug product, can include, but is not limited to, providing the drug product and the ACT peptide into or onto the target tissue. “Administering” a composition may be accomplished by injection, topical administration, or by any method in combination with other known techniques.

Unless otherwise indicated, the term “skin” means that outer integument or covering of the body, consisting of the dermis and the epidermis and resting upon subcutaneous tissue.

As used herein, the term “therapeutic” means an agent utilized to treat, combat, ameliorate, prevent or improve an unwanted condition or disease of a patient. In part, embodiments of the present invention are directed to the treatment of scarring and delayed wound healing.

As used herein, the phrase “effective amount” or “therapeutically effective amount” refers to a nontoxic but sufficient amount of the ACT polypeptides used in the practice of the invention that is effective to achieve the desired effect, i.e., to treat acute and chronic wounds. The activity contemplated by the present methods includes both medical therapeutic and/or prophylactic treatment, as appropriate, including, for example, a reduction and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. The specific dose of a compound administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the specific polypeptide administered and the condition being treated. A therapeutically effective amount of compound of this invention is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective intracellular concentration and local concentration in the tissue.

Generally speaking, the term “tissue” refers to any aggregation of similarly specialized cells which are united in the performance of a particular function. As used herein, “tissue”, unless otherwise indicated, refers to tissue which includes elastin as part of its necessary structure and/or function.

The terms “treat,” “treated,” or “treating” as used herein refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, ameliorate the effects of or slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; reducing the severity of a symptom of a condition, disease or disorder; reducing the frequency of a symptom of a condition, disease or disorder; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. In some embodiments, treatment may include preventing or reducing scarring and/or promoting the regeneration of complex tissue structure and function following injury.

As used herein, the terms “peptide,” “polypeptide,” or “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise the sequence of a protein or peptide. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs and fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides or a combination thereof. A peptide that is not cyclic will have an N-terminal and a C-terminal. The N-terminal will have an amino group, which may be free (i.e., as a NH2 group) or appropriately protected (for example, with a BOC or aFmoc group). The C-terminal will have a carboxylic group, which may be free (i.e., as a COOH group) or appropriately protected (for example, as a benzyl or a methyl ester). A cyclic peptide does not have free N- or C-terminal, since the ends are covalently bonded through an amide bond to form the cyclic structure. Amino acids may be represented by their full names (for example, leucine), 3-letter abbreviations (for example, Leu) and 1-letter abbreviations (for example, L). The structure of amino acids and their abbreviations can be found in the chemical literature, such as in Stryer, “Biochemistry”, 3rd Ed., W. H. Freeman and Co., New York, 1988.

Excipients for use in topical gels are well-known in the art and examples may be found in the Handbook of Pharmaceutical Excipients (Rowe, R. C. et al, APhA Publications; 5^(th) ed., 2005). Exemplary excipients may include waxes, various sugars and types of starch, polymers, gels, emollients, thickening agents, rheology modifiers, humectants, glycerol, organic basic compounds, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and solvents. Examples of rheology modifiers include Carbopol, hydroxypropyl cellulose, C₂₆₋₂₈ alkyl dimethicone, C₂₆₋₂₈ alkyl methicone, polyphenylsisquioxane, trimethylsiloxysilicate, crosspolymers of cyclopentasiloxane and dimethicone/vinyltrimethylsiloxysilicate, fumed silica (e.g. Cab-O-Sil M5P), and mixtures thereof. Examples of emollients include glycerine, pentylene glycol, sodium pyrrolidone carboxylic acid, lanolin, saccharide isomerate, stearoxy dimethicone, stearyl dimethicone, and mixtures thereof. Emollients may be useful to prevent stratum corneum dehydration occurring due to the use of anhydrous solvents in the formulation. Examples of organic bases include 2-amino-2-methyl propanol, niacinamide, methanolamines, triethanolamines, Trisamino, AMP-95, AmP-Ultra PC 2000, triisopropanolamine, diisopropanolamine, Neutrol TE, Ethomeen, and mixtures thereof. The organic base may render the pH of the medicament basic or neutral.

Other exemplary excipients include water-soluble porogens. A water-soluble porogen is an additive that may facilitate water uptake and diffusion into the gel. Any suitable porogen may be used, but in some embodiments, the porogen may include sodium chloride, potassium chloride, sucrose, glucose, lactose, sorbitol, xylitol, polyethylene glycol, polyvinylpyrrollidone, polyvinyl alcohol or mixtures thereof.

Polymers may also act as excipients in topical gels. Exemplary polymers include hydrophilic polyurethanes, hydrophilic polyacrylates, co-polymers of carboxymethylcellulose and acrylic acid, N-vinylpyrrolidone, poly(hydroxy acids), polyanhydrides, polyorthoesters, polyamides, polycarbonates, polyalkylenes (e.g., polyethylene and polypropylene), polyalkylene glycols (e.g., poly(ethylene glycol)), polyalkylene oxides (e.g., polyethylene oxide), polyalkylene terephthalates (e.g., polyethylene terephthalate), polyvinyl alcohols, polyvinyl ethers, polylvinyl esters, polyvinyl halides (e.g., poly(vinyl chloride)), polyvinylpyrrolidone, polysiloxanes, poly(vinyl acetates), polystyrenes, polyurethane copolymers, cellulose, derivatized celluloses (e.g., hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methylcellulose, ethylcellulose, carboxymethyl cellulose, or cellulose acetate), alginates, poly(acrylic acid), poly(acrylic acid) derivatives, acrylic acid copolymers, methacrylic acid, methacrylic acid derivatives, methacrylic acid copolymers, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), copolymers thereof and blends thereof.

In some embodiments of the invention, the polymers may be superabsorbent polymers (SAPs). A polymer is considered superabsorbent, as defined per IUPAC, as a polymer that can absorb and retain extremely large amounts of water relative to its own mass. SAPs may absorb water up to 500 times their own weight and may swell up to 1000-times their original volume. Particular SAPs of interest include sodium polyacrylate, the polyurethane Tecophilic TG-2000, and polymers prepared by the use of polyacrylamide copolymer, ethylene maleic anhydride copolymer, cross-linked carboxy-methyl-cellulose, polyvinyl alcohol copolymers, polyvinylpyrrolindone and cross-linked polyethylene oxide.

In some embodiments of the invention, polymers that are relatively hydrophobic may be used. Any suitable hydrophobic polymer may be used. However, exemplary polymers that are relatively hydrophobic include aromatic polyurethanes, silicone rubber, polysiloxanes, polycaprolactone, polycarbonate, polyvinylchloride, polyethylene, poly-L-lactide, poly-DL-glycolide, polyetheretherketone (PEEK), polyamide, polyimide and polyvinyl acetate. In addition, a hydrophobic gel-base and/or rheology modifier may be used.

In some embodiments of the invention, the polymers may act as thickening agents in the medicaments. Specifically, the polymeric portion of the gel may act as a visco-elastic substance and may retain the gel at the site of application, along with the alpha connexin polypeptides dispersed therein.

In some other embodiments, a gel that includes a polymer may have spreadability such that it forms a thin film when applied on the skin surface. This film may enable the application of the contained alpha connexin polypeptides over a wide area, and may serve to maintain the alpha connexin polypeptides on the affected area of the skin.

Other excipients may include various ionic or non-ionic compounds to maintain stability of the formulation, thereby protecting from the de-emulsification, settling, agglomeration or degradation of the formulation constituents that may reduce its therapeutic or aesthetic value.

Examples of ionic compounds may include salts such as sodium chloride, potassium chloride; cationic, anionic or zwitterionic surfactants such as sodium dodecyl sulfate (SDS), perfluorooctanoate (PFOA), perfluorooctanesulfonate (PFOS), ammonium lauryl sulfate (ALS), sodium lauryl ether sulfate (SLES), alkyl benzene sulfonate, cetyl trimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzethonium chloride, dodecyl betaine, cocamidopropyl betaine and cocoampho glycinate.

Examples of non-ionic compounds that may act as excipients include non-ionic surfactants such as Pluronic, Tween, AMP, and Brij family of surfactants; and surfactants derived from biological sources, e.g, natural or semi-synthetic surfactants, such as oleic acid, sorbitan trioleate, sorbitan monooleate, lecithin, cocamide MEA, cocamide DEA and cocamidopropyl betaine. Surfactants (both ionic and non-ionic) may reduce the interfacial surface energy and may facilitate spreading of the topical formulation over a wider area.

In some embodiments of the invention, solvent excipients may be used as a carrier vehicle for the alpha connexin polypeptides and other excipients. The polymer chains may interact with the solvent and undergo swelling to form a network that may impart visco-elastic properties to the topical formulation. In some embodiments of the topical formulation, the solvent may evaporate upon application, leaving a residual film of the polymer along with the entrapped alpha connexin polypeptides.

Exemplary solvent excipients that may be useful in hydrophilic formulations may include dimethyl isosorbide, propylene glycol, glycerol, isopropanol, ethanol, benzyl alcohol, ethylene glycol, polyethylene glycol, ethoxydiglycol or mixtures thereof. Exemplary solvent excipients that may be useful in hydrophobic formulations may include capric/caprylic triglycerides, isopropyl myristate, mineral oil, isododecane, isodecyl neopentanoate, butylene glycol, pentylene glycol, hexylene glycol, methoxypolyethyleneglycol, cyclopentasiloxane, cyclotetrasiloxane, dimethicone, caprylyl methicone or mixtures thereof.

In addition to the alpha connexin polypeptides and excipients, the topical formulation may also include at least one additional therapeutic agent such as antimicrobial agents, anti-acne agents, anti-inflammatory agents, analgesic agents, anesthetic agents, antihistamine agents, antiseptic agents, immunosuppressants, antihemorrhagic agents, vasodilators, wound healing agents, anti-biofilm agents and mixtures thereof.

Examples of antimicrobial agents include penicillins and related drugs, carbapenems, cephalosporins and related drugs, erythromycin, aminoglycosides, bacitracin, gramicidin, mupirocin, chloramphenicol, thiamphenicol, fusidate sodium, lincomycin, clindamycin, macrolides, novobiocin, polymyxins, rifamycins, spectinomysin, tetracyclines, vanomycin, teicoplanin, streptogramins, anti-folate agents including sulfonamides, trimethoprim and its combinations and pyrimethamine, synthetic antibacterials including nitrofurans, methenamine mandelate and methenamine hippurate, nitroimidazoles, quinolones, fluoroquinolones, isoniazid, ethambutol, pyrazinamide, para-aminosalicylic acid (PAS), cycloserine, capreomycin, ethionamide, prothionamide, thiacetazone, viomycin, eveminomycin, glycopeptide, glyclyclycline, ketolides, oxazolidinone; imipenen, amikacin, netilmicin, fosfomycin, gentamycin, ceftriaxone, Ziracin, Linezolid, Synercid, Aztreonam, and Metronidazole, Epiroprim, Sanfetrinem sodium, Biapenem, Dynemicin, Cefluprenam, Cefoselis, Sanfetrinem celexetil, Cefpirome, Mersacidin, Rifalazil, Kosan, Lenapenem, Veneprim, Sulopenem, ritipenam acoxyl, Cyclothialidine, micacocidin A, carumonam, Cefozopran and Cefetamet pivoxil.

Examples of topical anti-acne agents include adapalene, azelaic acid, benzoyl peroxide, clindamycin and clindamycin phosphate, doxycycline, erythromycin, keratolytics such as salicylic acid and retinoic acid (Retin-A”), norgestimate, organic peroxides, retinoids such as isotretinoin and tretinoin, sulfacetamide sodium, and tazarotene. Particular anti-acne agents include adapalene, azelaic acid, benzoyl peroxide, clindamycin {e.g., clindamycin phosphate), doxycycline {e.g., doxycycline monohydrate), erythromycin, isotretinoin, norgestimate, sulfacetamide sodium, tazarotene, etretinate and acetretin.

Examples of antihistamine agents include diphenhydramine hydrochloride, diphenhydramine salicylate, diphenhydramine, chlorpheniramine hydrochloride, chlorpheniramine maleate isothipendyl hydrochloride, tripelennamine hydrochloride, promethazine hydrochloride, methdilazine hydrochloride, and the like. Examples of local anesthetic agents include dibucaine hydrochloride, dibucaine, lidocaine hydrochloride, lidocaine, benzocaine, p-buthylaminobenzoic acid 2-(die-ethylamino) ethyl ester hydrochloride, procaine hydrochloride, tetracaine, tetracaine hydrochloride, chloroprocaine hydrochloride, oxyprocaine hydrochloride, mepivacaine, cocaine hydrochloride, piperocaine hydrochloride, dyclonine and dyclonine hydrochloride.

Examples of antiseptic agents include alcohols, quaternary ammonium compounds, boric acid, chlorhexidine and chlorhexidine derivatives, iodine, phenols, terpenes, bactericides, disinfectants including thimerosal, phenol, thymol, benzalkonium chloride, benzethonium chloride, chlorhexidine, povidone iode, cetylpyridinium chloride, eugenol and trimethylammonium bromide.

Examples of anti-inflammatory agents include nonsteroidal antiinflammatory agents (NSAIDs); propionic acid derivatives such as ibuprofen and naproxen; acetic acid derivatives such as indomethacin; enolic acid derivatives such as meloxicam, acetaminophen; methyl salicylate; monoglycol salicylate; aspirin; mefenamic acid; flufenamic acid; indomethacin; diclofenac; alclofenac; diclofenac sodium; ibuprofen; ketoprofen; naproxen; pranoprofen; fenoprofen; sulindac; fenclofenac; clidanac; flurbiprofen; fentiazac; bufexamac; piroxicam; phenylbutazone; oxyphenbutazone; clofezone; pentazocine; mepirizole; tiaramide hydrochloride; steroids such as clobetasol propionate, bethamethasone dipropionate, halbetasol proprionate, diflorasone diacetate, fluocinonide, halcinonide, amcinonide, desoximetasone, triamcinolone acetonide, mometasone furoate, fluticasone proprionate, betamethasone diproprionate, triamcinolone acetonide, fluticasone propionate, desonide, fluocinolone acetonide, hydrocortisone vlaerate, prednicarbate, triamcinolone acetonide, fluocinolone acetonide, hydrocortisone and others known in the art, predonisolone, dexamethasone, fluocinolone acetonide, hydrocortisone acetate, predonisolone acetate, methylpredonisolone, dexamethasone acetate, betamethasone, betamethasone valerate, flumetasone, fluorometholone, beclomethasone diproprionate, fluocinonide, topical corticosteroids, and may be one of the lower potency corticosteroids such as hydrocortisone, hydrocortisone-21-monoesters (e.g., hydrocortisone-21-acetate, hydrocortisone-21-butyrate, hydrocortisone-21-propionate, hydrocortisone-21-valerate, etc.), hydrocortisone-17,21-diesters (e.g., hydrocortisone-17,21-diacetate, hydrocortisone-17-acetate-21-butyrate, hydrocortisone-17,21-dibutyrate, etc.), alclometasone, dexamethasone, flumethasone, prednisolone, or methylprednisolone, or may be a higher potency corticosteroid such as clobetasol propionate, betamethasone benzoate, betamethasone dipropionate, diflorasone diacetate, fluocinonide, mometasone furoate, triamcinolone acetonide.

Examples of analgesic agents include alfentanil, benzocaine, buprenorphine, butorphanol, butamben, capsaicin, clonidine, codeine, dibucaine, enkephalin, fentanyl, hydrocodone, hydromorphone, indomethacin, lidocaine, levorphanol, meperidine, methadone, morphine, nicomorphine, opium, oxybuprocaine, oxycodone, oxymorphone, pentazocine, pramoxine, proparacaine, propoxyphene, proxymetacaine, sufentanil, tetracaine and tramadol.

Examples of anesthetic agents include alcohols such as phenol; benzyl benzoate; calamine; chloroxylenol; dyclonine; ketamine; menthol; pramoxine; resorcinol; troclosan; procaine drugs such as benzocaine, bupivacaine, chloroprocaine; cinchocaine; cocaine; dexivacaine; diamocaine; dibucaine; etidocaine; hexylcaine; levobupivacaine; lidocaine; mepivacaine; oxethazaine; prilocalne; procaine; proparacaine; propoxycaine; pyrrocaine; risocaine; rodocaine; ropivacaine; tetracaine; and derivatives, such as pharmaceutically acceptable salts and esters including bupivacaine HCl, chloroprocaine HCl, diamocaine cyclamate, dibucaine HCl, dyclonine HCl, etidocaine HCl, levobupivacaine HCl, lidocaine HCl, mepivacaine HCl, pramoxine HCl, prilocalne HCl, procaine HCl, proparacaine HCl, propoxycaine HCl, ropivacaine HCl, and tetracaine HCl.

Examples of antihemorrhagic agents include thrombin, phytonadione, protamine sulfate, aminocaproic acid, tranexamic acid, carbazochrome, carbaxochrome sodium sulfanate, rutin and hesperidin.

Beside the bioactive polypeptide component, the instant invention may also contain other active agents such as niacinamide, phytantriol, farnesol, bisabolol and salicylic acid. It is expected that certain additional active agents will act synergistically with the bioactive peptide component, or will enhance the shelf-life of the formulation.

Examples of wound treatments that may be used together with the drug product of the present invention include fibrinolytic enzymes, such as fibrinolysin, deoxyribonuclease, streptokinase, and streptodornase, necrotomy tissue agents containing lysozyme chloride, antimicrobial agents containing gentamicin sulfate, sulfadiazine silver, bacitracin, and fradiomycin sulfate, incarnant agents containing trafermin, bucladesine sodium, tretinoin tocoferil (tocoretinate), alprostadil alfadex, solcoseryl (extract from hemolysed blood of young cattle), and alcloxa, iodine preparations containing white soft sugar, povidone iodine, and iodine, and preparations containing bendazac, dimethyl isopropylazulene (guaiazulene), and epinephrine as active ingredients.

In addition to the alpha connexin polypeptides, excipients, and other therapeutic agents, the gels may also include other compounds that improve the organoleptic properties of the topical formulation.

Examples of such compounds include perfumes, dyes and colorants; chelating agents including but not limited to edetate disodium (EDTA), EGTA, CP94, citric acid; preservatives including but not limited to quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, cetrimide, dequalinium chloride, and cetylpyridinium chloride; mercurial agents, such as phenylmercuric nitrate, phenylmercuric acetate, and thimerosal; alcoholic agents, for example, chlorobutanol, phenylethyl alcohol, and benzyl alcohol; antibacterial esters, for example, esters of parahydroxybenzoic acid; and other anti-microbial agents such as chlorhexidine, chlorocresol, benzoic acid and polymyxin.

Alpha connexin polypeptides that may be used in the formulation of the present invention are described in U.S. Pat. No. 7,786,074, which is hereby incorporated by reference. In some embodiments, the alpha connexin polypeptide is a full length alpha connexin protein such as connexin 37, connexin 40, connexin 43, or connexin 45. In some embodiments, the polypeptide does not comprise the full-length connexin protein.

Peptides useful in the formulations of the present invention include alpha connexin polypeptides. The alpha connexin polypeptides useful in the formulations of the present invention may comprise or consist of the carboxy-terminal most 4 to 30 contiguous amino acids of an alpha connexin protein or conservative variant thereof. Thus, the polypeptides useful in the formulations can comprise the c-terminal-most 4 to 30 amino acids of the alpha Connexin, including the c-terminal most 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids of the alpha Connexin. In some aspects, the provided polypeptide further comprises a deletion of one or more amino acids of the c-terminal-most 4 to 30 amino acids of the alpha Connexin, including a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the c-terminal-most 4 to 30 amino acids of the alpha Connexin. For example, in some aspects, the polypeptides useful in the formulations of the present invention do not comprise the c-terminal-most 1, 2, or 3 amino acids of the alpha Connexin. For example, the polypeptides can consist essentially of the amino acid sequence SEQ ID NO:92, or a carboxy terminal fragment thereof of at least 4, 5, 6, 7, 8, 9, 10 amino acids in length.

In certain embodiments, the alpha connexin polypeptide of the present invention is linked at its amino or carboxy terminus to a cellular internalization transporter. Separate embodiments include the alpha connexin polypeptide without any cellular internalization transporter attached. The cellular internalization transporter linked to the alpha connexin polypeptides may be any internalization sequence known or newly discovered in the art, or conservative variants thereof. Non-limiting examples of cellular internalization transporters and sequences include Antennapedia sequences, TAT, HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II, Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (Bis-Guanidinium-Spermidine-Cholesterol, and BGTC (BisGuanidinium-Tren-Cholesterol).

In one embodiment of the present invention, the amino acid sequence of the alpha connexin polypeptide is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, and 5, or a conservative variant thereof. In a further embodiment of the present invention, the alpha connexin polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment of the present invention, the polypeptide comprises an alpha connexin polypeptide and a cellular internalization transporter. In a further embodiment, the alpha connexin polypeptide is linked at is amino terminus to the cellular internalization transporter. In a yet further embodiment, the amino acid sequence of the alpha connexin polypeptide linked to the cellular internalization transporter is selected from the group consisting of SEQ ID NO: 8, 9, 10, 11, and 12, or a conservative variant thereof. In one embodiment, the polypeptide consists of or comprises the amino acid sequence of SEQ ID NO: 9, namely, RQPKIWFPNRRKPWKKRPRPDDLEI.

When specific proteins are referred to herein, variants, derivatives, and fragments are contemplated. Protein variants and derivatives are well understood to those of skill in the art and can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known and include, for example, M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues. Deletions or insertions may be made in adjacent pairs, i.e., a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure unless such a change in secondary structure of the mRNA is desired. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are referred to as conservative substitutions. For example, the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions Conservatively substituted variations of each explicitly disclosed sequence are included within the polypeptides provided herein. Typically, conservative substitutions have little to no impact on the biological activity of a resulting polypeptide. In a particular example, a conservative substitution is an amino acid substitution in a peptide that does not substantially affect the biological function of the peptide. A peptide can include one or more amino acid substitutions, for example 2-10 conservative substitutions, 2-5 conservative substitutions, 4-9 conservative substitutions, such as 2, 5 or 10 conservative substitutions. A polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR. Alternatively, a polypeptide can be produced to contain one or more conservative substitutions by using standard peptide synthesis methods. An alanine scan can be used to identify which amino acid residues in a protein can tolerate an amino acid substitution. In one example, the biological activity of the protein is not decreased by more than 25%, for example not more than 20%, for example not more than 10%, when an alanine, or other conservative amino acid (such as those listed below), is substituted for one or more native amino acids. Further information about conservative substitutions can be found in, among other locations, in Ben-Bassat et al., (J. Bacterial. 169:751-7, 1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al., (Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321-5, 1988) and in standard textbooks of genetics and molecular biology.

As used herein, “analytical methods” includes the following techniques that are useful for determining the identity and quantity of the ACT peptide in a particular formulation: NMR, HPLC, Amino Acid Composition and Molecular Weight Determinations, and Specific Optical Rotation.

The present invention includes a topical formulation comprising at least one alpha connexin polypeptide and hydroxyethylcellulose gel, wherein the hydroxyethylcellulose gel stabilizes the alpha connexin polypeptide. In certain embodiments, the hydroxyethylcellulose gel stabilizes the alpha connexin polypeptide so that after 3 months of storage at 5° C. at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the alpha connexin polypeptide is detectable by analytical methods. In some embodiments, the alpha connexin polypeptide is present in the formulation at a concentration of about 0.0025% (w/w), of about 0.005% (w/w), of about 0.0075% (w/w), of about 0.010% (w/w), of about 0.015% (w/w), of about 0.020% (w/w), of about 0.025% (w/w), of about 0.030% (w/w), of about 0.035% (w/w), of about 0.040% (w/w), of about 0.045% (w/w), of about 0.050% (w/w), of about 0.055% (w/w), of about 0.060% (w/w), of about 0.065% (w/w), of about 0.070% (w/w), of about 0.075% (w/w), of about 0.080% (w/w), of about 0.085% (w/w), of about 0.090% (w/w), of about 0.095% (w/w), of about 0.100% (w/w), of about 0.150% (w/w), of about 0.200% (w/w), of about 0.250% (w/w), of about 0.500% (w/w), of about 0.750% (w/w), of about 1.00% (w/w), of about 1.50% (w/w), of about 2.00% (w/w), of about 2.50% (w/w), or of about 5.00 (w/w). In one embodiment, the alpha connexin polypeptide is present in the formulation at a concentration of between about 0.005% (w/w) and about 1.00% (w/w).

In other embodiments, the drug product of the invention is a clear colorless gel which contains 0.0072% (w/w) (20 μM) of the ACT peptide, 0.018% (w/w) (50 μM) of the ACT peptide, 0.036% (w/w) (100 μM) of the ACT peptide, or 0.072% (w/w) (200 μM) of the ACT peptide. The ACT peptide may be dissolved in a semisolid dosage form that contains >0% water, >10% water, >20% water, >30% water, >40% water, >50% water, >60% water, >70% water, >80% water, or >90% water and 0.25% gelling agent (polymer), 0.55% gelling agent (polymer), 0.75% gelling agent (polymer), 1.00% gelling agent (polymer), 1.25% gelling agent (polymer), 1.50% gelling agent (polymer), 1.75% gelling agent (polymer), 2.00% gelling agent (polymer), 2.25% gelling agent (polymer), or 2.50% gelling agent (polymer). The ACT peptide may be well preserved and adequately buffered to pH 6. In one embodiment of the topical formulation, the qualitative and quantitative composition is that listed in Table 1.

TABLE 1 Drug Product Gel Qualitative & Quantitative Composition Concentration Ingredients Grade Function (% w/w) Peptide 328967 — Active 0.0072; 0.018 (ACT peptide) 0.036; 0.072 Methylparaben NF Preservative 0.17 Propylparaben NF Preservative 0.02 Glycerin USP Solvent 5.0 Sodium Phosphate USP Buffer Agent 0.263 Monobasic Sodium Phosphate Dibasic USP Buffer Agent 0.044 Propylene Glycol USP Solvent 3.0 Edetate Disodium (EDTA) USP Chelating Agent 0.05 D-Mannitol USP Stabilizer 0.05 Hydroxyethylcellulose, NF Gelling Agent 1.25% 250HHX Purified Water, qsad USP Solvent  100%

The ACT1 peptide sequence is listed in Table 2 below in which Ahx refers to L-2-aminohexanoic acid, also known as 6-aminohexanoic acid:

TABLE 2 Peptide 328967 (Antp/ACT1) sequence. Biotin-Ahx-Arg-Gln-Pro-Lys-Ile-Trp-Phe-Pro-Asn- Arg-Arg-Lys-Pro-Trp-Lys-Lys-Arg-Pro-Arg-Pro-Asp- Asp-Leu-Glu-Ile-OH

The general properties of the Peptide 328967 are listed in Table 3.

TABLE 3 General Physical Properties of Peptide 328967 Physical Appearance White to off-white powder Molecular Weight 3597.33 ± 2.0 amu Counter-Ion AcOH Solubility Soluble in water at room temperature

In one aspect of the invention, the excipients used in the drug product topical preparation are selected from the group consisting of or are one or more of the following:

Methylparaben

Propylparaben

Glycerin

Sodium Phosphate Monobasic

Sodium Phosphate Dibasic

Propylene Glycol

Edetate Disodium (EDTA)

D-Mannitol

Hydroxyethylcellulose, 250 HHX

Purified Water

In one embodiment, the drug product topical preparation comprises a peptide, D-mannitol, hydroxyethylcellulose, and purified water. Said preparation may further comprise one or more of methylparaben, propylparaben, glycerin, sodium phosphate monobasic, sodium phosphate dibasic, propylene glycol, and edetate disodium (EDTA). In a further embodiment, the hydroxyethylcellulose is 250 HHX. In a further embodiment, the peptide is an alpha connexin polypeptide.

In such embodiments, the drug product topical preparation comprises a peptide at a concentration between about 0.001% (w/w) and about 0.5% (w/w) (for example, at about 0.0072%, 0.018%, 0.036%, or 0.072% (w/w)); methylparaben at a concentration between about 0.10% (w/w) and about 0.25% (w/w) (for example, about 0.17% (w/w)); propylparaben at a concentration between about 0.01% (w/w) and about 0.03% (w/w) (for example, about 0.02% (w/w)); glycerin at a concentration between about 1% (w/w) and about 10% w/w) (for example, about 5% (w/w)); sodium phosphate monobasic at a concentration between about 0.1% (w/w) and about 0.5% (w/w) (for example, about 0.263% (w/w)); sodium phosphate dibasic at a concentration between about 0.02% and about 0.06% (for example, about 0.044% (w/w)); propylene glycol at a concentration between about 1% (w/w) and about 5% (w/w) (for example, about 3% (w/w)); EDTA at a concentration between about 0.01% and about 0.1% (for example, about 0.05% (w/w)); D-mannitol at a concentration between about 0.01% (w/w) and about 0.1% (w/w) (for example, about 0.05% (w/w)); hydroxyethylcellulose at a concentration between about 0.5% and about 2.5% (for example, about 1.25% (w/w)), and purified water at a concentration of about 0.1% to about 10% (for example, about 1%). In a further embodiment, the peptide is an alpha connexin polypeptide. In a yet further embodiment, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 8, 9, 10, 11, and 12. In one embodiment, the peptide comprises an amino acid sequence according to SEQ ID NO: 9.

Derived through in vitro and in vivo studies, these stabilizers and excipients are incorporated into the drug product since they are non-irritating, non-staining, and non-immunogenic. Stability studies showed that the ACT1 peptide is more stable in the gelling agent Hydroxyethylcellulose, 250 HHX (1.25%) compared to Pluronic gels. The ACT peptide within the drug product containing 1.25% hydroxyethylcellulose only dropped to 98% of the label claim (i.e., initial concentration) when stored at 5° C. for three months and to 84% of the label claim when stored at 25° C. for the same duration. In one aspect of the invention, edetate disodium (EDTA) and Mannitol are incorporated within the drug product to provide stability to the ACT1 peptide. In some embodiments of the invention, the Mannitol is present in the formulation at 0.01% (w/w) to 1.6% (w/w), 0.01% (w/w) to 1.5% (w/w), 0.01% (w/w) to 1.4% (w/w), 0.01% (w/w) to 1.3% (w/w), 0.01% (w/w) to 1.2% (w/w), 0.01% (w/w) to 1.1% (w/w), 0.01% (w/w) to 1.0% (w/w), 0.01% (w/w) to 0.9% (w/w), 0.01% (w/w) to 0.8% (w/w), 0.01% (w/w) to 0.7% (w/w), 0.01% (w/w) to 0.6% (w/w), 0.01% (w/w) to 0.5% (w/w), 0.01% (w/w) to 0.4% (w/w), 0.01% (w/w) to 0.3% (w/w), 0.01% (w/w) to 0.2% (w/w), 0.01% (w/w) to 0.1% (w/w), or 0.01% (w/w) to 0.0.05% (w/w). In one embodiment, the Mannitol is present in the formulation at about 0.05% (w/w).

In another aspect of the present invention, a buffering agent is included in the topical formulation to maintain the pH in a certain range. Suitable buffering agents can include, but are not limited to, acetate buffers, citrate buffers, phosphate buffers, lactic acid buffers, malic acid buffers, succinic acid buffers, borate buffers, sodium hydroxide, potassium hydroxide, and ammonium hydroxide. Phosphate salts such as monosodium phosphate (NaH₂PO₄; also known as monobasic sodium phosphate), disodium hydrogen phosphate (Na₂HPO₄; also known as dibasic sodium phosphate), monopotassium phosphate (KH₂PO₄), dipotassium phosphate (K₂HPO₄), and mixtures thereof can also be used. In one embodiment, phosphate buffer provides superior stability compared to citrate buffer. A buffer capacity at 25 mM was found to be adequate for the drug product. Buffer is needed to control pH of the gel system and to maintain stability of the peptide drug. The pH range of the topical formulation may be pH 2 to pH 12, pH 4 to pH 10, or pH 6 to pH 8. In one embodiment, the proper pH range is in the range of 5 to 7. In some embodiments, the pH of the topical formulation of the present invention is 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. In yet another aspect, propylene glycol in the amount of 3% provides better solubilization of parabens in the aqueous system.

In one embodiment, the formulation with an ACT peptide incorporated into hydroxyethylcellulose enables large scale manufacturing of a product with the characteristics that make it practical for clinical treatments as well as meeting desired storage and stability requirements. While a formulation for ACT1 using Pluronic gel may require a relatively long incorporation time of approximately 2.5 hours and only yield 50 gram batches, a formulation with hydroxyethylcellulose provides allows for significantly faster incorporation of the ACT peptide into the gel and yields much larger batches. For example, when using Pluronic F 127 gel in the topical formulation it takes over an hour to incorporate the polymer, and the formulation needs to be placed in a water bath to help with incorporation. In contrast, hydroxyethylcellulose (e.g., HEC 250 HHX) is easily incorporated at room temperature and hydrates within 30 minutes. Thus, the use of hydroxyethylcellulose may facilitate large scale manufacturing. The manufacturing process with Pluronic gel may require a cold bath to bring its viscosity into a desired range and require more energy than the manufacturing process with hydroxyethylcellulose. In addition, the final formulation in Pluronic gel may be very thin at a storage condition of 5 degrees C.

It has been discovered that hydroxyethylcellulose (HEC) is a suitable gelling agent and acceptable carrier of the drug product of the present invention. In one embodiment, the gelling agent is Hydroxyethylcellulose (HEC), 250 HHX. In one embodiment, the percent (w/w) of HEC is in the range of 1-5%. In a further embodiment, the percent (w/w) of HEC is 1.25%. In the manufacture of HEC, a purified cellulose is reacted with sodium hydroxide to produce a swollen alkali cellulose. The alkali-treated cellulose is more chemically reactive than cellulose. By reacting the alkali cellulose with ethylene oxide, a series of hydroxyethylcellulose ethers is produced. In this reaction, the hydrogen atoms in the hydroxyl groups of cellulose are replaced by hydroxyethyl groups, which confer water solubility to the gel. It is contemplated in this invention that a single HEC ether may be used, or a mixture of HEC ethers of difference molecular weight and structure may be used. Suitable grades of HEC for pharmaceutical purposes are well known and full described in the pharmaceutical literature. Suitable commercially available brands of HEC include but are not limited to Fuji HEC-HP; Fuji HEC-AG 15; NATRO-SOL 250HR; NATROSOL 250 MH; NATROSOL 250G; CELLOSIZE QP 30000; TYLOSE H SERIES; NATROSOL 180L; NATROSOL 300H; TYLOSE P-X; NATROSOL 250M; CELLOSIZE WP 4400; CELLOSIZE UT 40; NATROSOL 250H4R; Tylose H 20P; NATROSOL LR; TYLOSE MHB; NATROSOL 250HHP; HERCULES N 100; CELLOSIZE WP 300; TYLOSE P-Z SERIES; NATROSOL 250H; TYLOSE PS-X; Cellobond HEC 400; CELLOSIZE QP; CELLOSIZE QP 1500; NATRO-SOL 250; HYDROXYETHYL CELLULOSE ETHER; HESPAN; TYLOSE MHB-Y; NATROSOL 240JR; HYDROXYETHYL STARCH; CELLOSIZE WP; CELLOSIZE WP 300H; 2-HYDROXYETHYL CELLULOSE ETHER; BL 15; CELLOSIZE QP 4400; CELLOSIZE QP3; TYLOSE MB; CELLULOSE HYDROXY-ETHYLATE; CELLOSIZE WPO 9H17; CELLOSIZE 4400H16; CELLULOSE HYDROXYETHYL ETHER; Hydroxyethyl Cellulose; Hydroxyl Ethyl Cellulose (HEC); Hydroxyethyl Cellulose 100H (celocell 100h); TYLOSE MH-XP; NATROSOL 250HX; Natrosol; Daicel EP 500; HEC-Unicel; HEC (Hydroxyethyl cellulose); Cellosize; HEC-Al 5000; Fuji HEC-AL 15; HEC-Unicel QP 09L; Cellulose, ethers, 2-hydroxyethyl ether; Unicel QP 52000H; HEC-QP 4400; SP 250 (cellulose); Hetastarch; Cellulose, ethers, 2-hydroxyethyl ether; Glutofix 600; FL 52; Fuji HEC-AX 15F; Tylose H 300P; HEC-Unicel QP 300H; Tylose H 300; Daicel SP 550; Daicel SE 600; Unicel QP 15000; HEC-QP 100 MH; HEC-QP 9H; OETs; Daicel EP 850; H. E. Cellulose; Cellobond 25T; Unicel QP 100 MH; Tylose H 4000; SE 850K; Tylomer H 20; Daicel SE 850K; Tylose H 30000YP; Unicel QP 4400; SP 407; Tylose H 100000; Daicel SP 200; Culminal HEC 5000PR; Tylopur H 300; Daicel SP 750; Sanhec; BL 15 (cellulose derivative); Unicel QP 300H; Tylomer H 200; J 164; Tylose H 10; Tylose H 20; AH 15; Daicel SP 600; Daicel SE 900; HEC-Unicel QP 4400H; AX 15; Daicel SP 800; Fuji HEC-AW 15F; HEC-SE 850; HEC-A 5-25CF; Metolose 90SEW; AW 15 (polysaccharide); Cellobond HEC 5000; HEC-QP 100M; Cellobond HEC 15A; Tylose H 15000YP2; Walocel HT 6.000 PFV; 2-Hydroxyethyl cellulose (Natrosol Type 250HRCS); Fuji HEC-BL 20; Fuji HEC-SY 25F; Telhec; HEC-SP 200; HEC-AH 15; HEC-Unicel QP 30000H; see; HEC 10A; Daicel SP 400; Admiral 3089FS; Fuji HEC-A 5000F; HEC-SP 400; Hydroxyethyl Methyl Cellulose (HEMC); HYDROXYETHYL CELLULOSE (HEC); Hydroxyethyl Starch (CAS No:9004-62-0); Hydroxy Ethyl Cellulose; “Natrosol” [Aqualon]; HEC; 2-HYDROXYETHYL CELLULOSE; NATROSOL 150L; TYLOSE MHB-YP; HYDROXYETHYL ETHER CELLULOSE; NATROSOL 250L; CELLOSIZE WP 400H; TYLOSE P; CELLULOSE, 2-HYDROXYETHYL ETHER; TYLOSE MH-K; NATROSOL 250HHR.

In another embodiment, the drug product of the present invention is packaged in 20 mL vial (USP Type I, Borosilicate clear scintillation glass with poly-seal cone urea screw cap). A mixture of methylparaben at 0.17% (w/w) and propylparaben 0.02% (w/w) is used as a preservative.

In some embodiments, the present invention includes a method of wound treatment comprising administering to a subject in need thereof a topical formulation comprising at least one alpha connexin polypeptide and hydroxyethylcellulose gel, wherein the hydroxyethylcellulose gel stabilizes the alpha connexin polypeptide. The wound treated may be an acute surgical wound or a chronic, non-infected, full-thickness lower extremity ulcer.

In a certain embodiment, the drug product of the present invention may be used to mitigate excessive scar formation associated with acute surgical wounds. In this embodiment, the drug product of the present invention may be applied at the time of surgical incision closure, 1 hour after surgical incision closure, 2 hours after surgical incision closure, 3 hours after surgical incision closure, 4 hours after surgical incision closure, 5 hours after surgical incision closure, 6 hours after surgical incision closure, 7 hours after surgical incision closure, 8 hours after surgical incision closure, 9 hours after surgical incision closure, 10 hours after surgical incision closure, 11 hours after surgical incision closure, 12 hours after surgical incision closure, 13 hours after surgical incision closure, 14 hours after surgical incision closure, 15 hours after surgical incision closure, 16 hours after surgical incision closure, 17 hours after surgical incision closure, 18 hours after surgical incision closure, 19 hours after surgical incision closure, 20 hours after surgical incision closure, 21 hours after surgical incision closure, 22 hours after surgical incision closure, 23 hours after surgical incision closure, 24 hours after surgical incision closure, 48 hours after surgical incision closure, 72 hours after surgical incision closure, or thereafter.

In another embodiment, the drug product of the present invention may be used to treat chronic ulcers. For example, ulcers may include diabetic foot ulcers, venous leg ulcers, and pressure ulcers. These ulcers may be chronic, non-infected, full-thickness lower extremity ulcers. In one embodiment, the drug product of the present invention may be applied to a chronic ulcer in a daily regimen, a regimen of every other day, a regimen of once a week, or in various other regimens until healing of the chronic ulcer is apparent. In another embodiment, the drug product of the present invention may be applied to a chronic ulcer in a regimen at day 0, 3, 7, 14, 21, and 28. In another embodiment, the drug product of the present invention may be applied to a chronic ulcer in a regimen at day 0, day 3, week 1, week 2, week 3, week 4, week 5, week 6, week 7, week 8, week 9, week 10, week 11, and week 12. In another aspect of the present invention, the drug product is manufactured with the following steps:

Step 1: In a suitable size of beaker, add propylene glycol, glycerin, methylparaben and propylparaben. Mix with a propeller until the parabens are completely dissolved.

Step 2: In a manufacturing vessel, add purified water (part I), EDTA, monobasic sodium phosphate, dibasic sodium phosphate and D-mannitol. Mix with a propeller until a clear solution is obtained.

Step 3: Add the solution from step 1 to the manufacturing vessel. Rinse the beaker with purified water (part II, divided into approximately 3 equal portions) and add the rinse back to the vessel. Continue with propeller mixing until the solution is visually homogeneous.

Step 4: With homogenization mixing, add hydroxyethyl cellulose into the manufacturing vessel from Step 3. Mix until the polymer is fully dispersed.

Step 5: In a separate beaker, add purified water (part III) and an alpha connexin polypeptide (e.g., Peptide 328967 ACT1 peptide). Mix with a stir bar or propeller mixer until the peptide is completely dissolved and a gel is formed.

Step 6: With continuous propeller mixing, add the drug solution from step 5 to the manufacturing vessel. Rinse the beaker with purified water (part IV, divided into approximately 3 equal portions) and add the rinse back to the vessel. Mix until the gel is homogeneous.

The manufacturing process flow chart is provided in FIG. 1 Manufacturing Process Flow Chart.

Sequences

SEQ ID NO: 1 (ACT 2) PSSRASSRASSRPRPDDLEI SEQ ID NO: 2 (ACT 1) RPRPDDLEI SEQ ID NO: 3 (ACT 3) RPRPDDLEV SEQ ID NO: 4 (ACT 4) RPRPDDVPV SEQ ID NO: 5 (ACT 5) KARSDDLSV SEQ ID NO: 6 aga cct cgg cct gat gac ctg gag att SEQ ID NO: 7 (Antp) RQPKIWFPNRRKPWKK SEQ ID NO: 8 (Antp/ACT 2) RQPKIWFPNRRKPWKKPSSRASSRASSRPRPDDLEI SEQ ID NO: 9 (Antp/ACT 1) RQPKIWFPNRRKPWKKRPRPDDLEI SEQ ID NO: 10 (Antp/ACT 3) RQPKIWFPNRRKPWKKRPRPDDLEV SEQ ID NO: 11 (Antp/ACT 4) RQPKIWFPNRRKPWKKRPRPDDVPV SEQ ID NO: 12 (Antp/ACT 5) RQPKIWFPNRRKPWKKKARSDDLSV SEQ ID NO: 13 (encodes polypeptide of SEQ ID NO 9) cgg cag ccc aag atc tgg ttc ccc aac cgg cgg aag ccc tgg aag aag cgg ccc ggc ccg acg acc tgg aga tc SEQ ID NO: 14 (HIV-Tat) GRKKRRQRPPQ SEQ ID NO: 15 (Penetratin) RQIKIWFQNRRMKWKK SEQ ID NO: 16 (Antp-3A) RQIAIWFQNRRMKWAA SEQ ID NO: 17 (Tat) RKKRRQRRR SEQ ID NO: 18 (Buforin II) TRSSRAGLQFPVGRVHRLLRK SEQ ID NO: 19 (Transportan) GWTLNSAGYLLGKINKALAALAKKIL SEQ ID NO: 20 (model amphipathic peptide) KLALKLALKALKAALKLA SEQ ID NO: 21 (K-FGF) AAVALLPAVLLALLAP SEQ ID NO: 22 (Ku70) VPMLK-PMLKE SEQ ID NO: 23 (Prion) MANLGYWLLALFVTMWTDVGLCICICRPKP SEQ ID NO: 24 (pVEC) LLIILRRRIRKQAHAHSK SEQ ID NO: 25 (Pep-1) KETWWETWWTEWSQPKKKRKV SEQ ID NO: 26 (SynB1) RGGRLSYSRRRFSTSTGR SEQ ID NO: 27 (Pep-7) SDLWEMMMVSLACQY SEQ ID NO: 28 (HN-1) TSPLNIHNGQKL SEQ ID NO: 29 (Chick alpha Cx43 ACT) PSRASSRASSRPRPDDLEI SEQ ID NO: 30 (Human alpha Cx45) GSNKSTASSKSPDPKNSVWI SEQ ID NO: 31 (Chick alpha Cx45) GSNKSSASSKSGDGKNSVWI SEQ ID: 32 (Human alpha Cx46) GRASKASRASSGRARPEDLAI SEQ ID: 33 (Human alpha Cx46.6) GSASSRDGKTVWI SEQ ID NO: 34 (Chimp alpha Cx36) PRVSVPNFGRTQSSDSAYV SEQ ID NO: 35 (Chick alpha Cx36) PRMSMPNFGRTQSSDSAYV SEQ ID NO: 36 (Human alpha Cx47) PRAGSEKGSASSRDGKTTVWI SEQ ID NO: 37 (Human alpha Cx40) GYHSDKRRLSKASSKARSDDLSV SEQ ID NO: 38 (Human alpha Cx50) PLSRLSKASSRARSDDLTV SEQ ID NO: 39 (Human alpha Cx59) PNHVVSLTNNLIGRRVPTDLQI SEQ ID NO: 40 (Rat alpha Cx33) PSCVSSSAVLTTICSSDQVVPVGLSSFYM SEQ ID NO: 41 (Sheep alpha Cx44) GRSSKASKSSGGRARAADLAI SEQ ID NO: 42 (Human beta Cx26) LCYLLIRYCSGKSKKPV SEQ ID: 43 (Human alpha Cx37) GQKPPSRPSSSASKKQ*YV SEQ ID 44: (conservative Cx43 variant) SSRASSRASSRPRPDDLEV SEQ ID 45: (conservative Cx43 variant) RPKPDDLEI SEQ ID 46: (conservative Cx43 variant) SSRASSRASSRPKPDDLEI, SEQ ID 47: (conservative Cx43 variant) RPKPDDLDI SEQ ID 48: (conservative Cx43 variant) SSRASSRASSRPRPDDLDI SEQ ID 49: (conservative Cx43 variant) SSRASTRASSRPRPDDLEI SEQ ID 50: (conservative Cx43 variant) RPRPEDLEI SEQ ID 51: (conservative Cx43 variant) SSRASSRASSRPRPEDLEI, SEQ ID 52: (conservative Cx45 variant) GDGKNSVWV SEQ ID 53: (conservative Cx45 variant) SKAGSNKSTASSKSGDGKNSVWV SEQ ID 54: (conservative Cx37 variant) GQKPPSRPSSSASKKLYV SEQ ID NO: 55 (non-active control peptide) RQPKIWFPNRRKPWKIELDDPRPR SEQ ID NO: 56 (HIV-Tat/ACT 1) GRKKRRQRPPQ RPRPDDLEI SEQ ID NO: 57 (Penetratin/ACT 1) RQIKIWFQNRRMKWKK RPRPDDLEI SEQ ID NO: 58 (Antp-3A/ACT 1) RQIAIWFQNRRMKWAA RPRPDDLEI SEQ ID NO: 59 (Tat/ACT 1) RKKRRQRRR RPRPDDLEI SEQ ID NO: 60 (Buforin II/ACT 1) TRSSRAGLQFPVGRVHRLLRK RPRPDDLEI SEQ ID NO: 61 (Transportan/ACT 1) GWTLNSAGYLLGKINKALAALAKKIL RPRPDDLEI SEQ ID NO: 62 (MAP/ACT 1) KLALKLALKALKAALKLARPRPDDLEI SEQ ID NO: 63 (K-FGF/ACT 1) AAVALLPAVLLALLAP RPRPDDLEI SEQ ID NO: 64 (Ku70/ACT 1) VPMLKPMLKERPRPDDLEI SEQ ID NO: 65(Prion/ACT 1) MANLGYWLLALFVTMWTDVGLCKKRPKPRPRPDDLEI SEQ ID NO: 66 (pVEC/ACT 1) LLIILRRRIRKQAHAHSK RPRPDDLEI SEQ ID NO: 67 (Pep-1/ACT 1) KETWWETWWTEWSQPKKKRKV RPRPDDLEI SEQ ID NO: 68 (SynB1/ACT 1) RGGRLSYSRRRFSTSTGR RPRPDDLEI SEQ ID NO: 69 (Pep-7/ACT 1) SDLWEMMMVSLACQY RPRPDDLEI SEQ ID NO: 70 (HN-1/ACT 1) TSPLNIHNGQKL RPRPDDLEI SEQ ID NO: 71 (20 to 120 residues flanking amino acid 363 of human Cx43) KGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSPPGYKLVT GDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDD NQNSKKLAAGHELQPLAIVDQR SEQ ID NO: 72 (20 to 120 residues flanking amino acid 362 of chick Cx43) KTDPYSHSGTMSPSKDCGSPKYAYYNGCSSPTAPLSPMSPPGYKLVTGDR NNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFADEHQN TKKLASGHELQPLTIVDQRP SEQ ID NO: 73 (20 to 120 residues flanking amino acid 377 of human Cx45) LGFGTIRDSLNSKRRELEDPGAYNYPFTWNTPSAPPGYNIAVKPDQIQYT ELSNAKIAYKQNKANTAQEQQYGSHEENLPADLEALQREIRMAQERLDLA VQAYSHQNNPHGPREKKAKV SEQ ID NO: 74 (20 to 120 residues flanking amino acid 375 of chick Cx45) GFGTIRDTLNNKRKELEDSGTYNYPFTWNTPSAPPGYNIAVKPDQMQYTE LSNAKMAYKQNKANIAQEQQYGSNEENIPADLENLQREIKVAQERLDMAI QAYNNQNNPGSSSREKKSKA. SEQ ID NO: 75 (20 to 120 residues flanking amino acid 313 of human Cx37) PYLVDCFVSRPTEKTIFIIFMLVVGLISLVLNLLELVHLLCRCLSRGMRA RQGQDAPPTQGTSSDPYTDQVFFYLPVGQGPSSPPCPTYNGLSSSEQNWA NLTTEERLASSRPPLFLDPP SEQ ID NO: 76 (20 to 120 residues flanking amino acid 258 of rat Cx33) CGSKEHGNRKMRGRLLLTYMASIFFKSVFEVAFLLIQWYLYGFTLSAVYI CEQSPCPHRVDCFLSRPTEKTIFILFMLVVSMVSFVLNVIELFYVLFKAI KNHLGNEKEEVYCNPVELQK. SEQ ID NO: 77 (enhanced green fluorescent protein) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIF FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH YLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 78 (ACT 2) CCCTCCTCCCGGGCCTCCTCCCGGGCCTCCTCCCGGCCCCGGCCCGACGA CCTGGAGATC SEQ ID NO: 79 (ACT 1) CGGCCCCGGCCCGACGACCTGGAGATC SEQ ID NO: 80 (ACT 3) CGGCCCCGGCCCGACGACCTGGAGGTG SEQ ID NO: 81 (ACT 4) CGGCCCCGGCCCGACGACGTGCCCGTG SEQ ID NO: 82 (ACT 5) AAGGCCCGGTCCGACGACCTGTCCGTG SEQ ID NO: 83 (Antp) CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAG AAG SEQ ID NO: 84 (Antp/ACT 2) CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCC CTCCTCCCGGGCCTCCTCCCGGGCCTCCTCCCGGCCCCGGCCCGACGACC TGGAGATC SEQ ID NO: 85 (Antp/ACT 1) CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCG GCCCCGGCCCGACGACCTGGAGATC SEQ ID NO: 86 (Antp/ACT 3) CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCG GCCCCGGCCCGACGACCTGGAGGTG SEQ ID NO: 87 (Antp/ACT 4) CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCG GCCCCGGCCCGACGACGTGCCCGTG SEQ ID NO: 88 (Antp/ACT 5) CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGAA GGCCCGGTCCGACGACCTGTCCGTG SEQ ID NO: 89 (Zebrafish alpha Cx43) PCSRASSRMSSRARPDDLDV SEQ ID NO: 90 (Chick alpha Cx36) PRVSVPNFGRTQSSDSAYV SEQ ID NO: 91 (Zebrafish alpha Cx36) PRMSMPNFGRTQSSDSAYV SEQ ID NO: 92 (Cx43 isoleucine deletion) RQPKIWFPNRRICPWICKRASSRASSRPRPDDLE

EXAMPLES Example 1

The objective was to develop a stable, elegant and pourable gel in which the Active Pharmaceutical Ingredient (API) is in solution for wound healing purposes.

The major challenges encountered during formulation development were the selection of suitable stabilizers for the API (ACT1), and other excipients that are non-irritating for wound healing application. Studies were conducted to evaluate prototype formulations.

R&D Study Per Protocol #1062

The first study was conducted per protocol #1062. During this study, 6 prototype formulations were evaluated as outlined in Table 4. The purposes of the study were to select a proper pH range for the product and also to select a proper gelling agent. The API concentration used for this study was 0.072% which was the initial targeted product concentration. Phosphate buffers at pH 5, 6, and 7 and two gelling agents, Pluronic and Hydroxyethylcellulose (HEC) were evaluated. The quantity of API required for the formulations was calculated as follows:

Batch size for each formulation=12×5 g=60 g or 70 g

API quantity for 6 formulations=6×70×0.072%/82.4%=0.37 g

API purity value=82.4%

Five formulations, formulae 2414-10 to -14, were gelled with Pluronic 127, which was assumed to be the preferred gelling agent. Only one formulation, 2414-15, was gelled with HEC as a back-up. The available stability data indicated the following conclusions.

Buffer was needed to control the pH of the gel system and to maintain stability of the peptide drug. Proper pH range seemed to be between 5 and 7. Phosphate buffer was adequate.

Surprisingly, the API, ACT1, was more stable in HEC gel than Pluronic gels. With Pluronic gels, the API dropped to an average of 85% of the label claim (LC) from initial when gels were stored at 5° C. for one month; and to 79% when stored at 25° C. for one month. With HEC gel, the API dropped to 98% of label claim from initial when stored at 5° C. for 3 months and 84% when stored at 25° C. for three months.

HEC appeared to be a better choice of gelling agent than Pluronic when comparing formula 2414-14 to 2414-15. Both formulations were exactly same except that formula 2414-14 was gelled with Pluronic and formula 2414-15 was gelled with HEC. The API in formula 2414-14 dropped to 68% of the label claim from the initial concentration when stored at 5° C. for one month while the API in formula 2414-15 only dropped to 98% of the label claim from the initial concentration when stored at 5° C. for 3 months.

EDTA and Mannitol appear to provide some stability to the peptide drug.

The formulations did not appear to be stable at 25° C. The final product might require refrigeration, which is typical for products containing peptides and proteins.

Formula 2414-15 with HEC as the gelling agent is the most stable formulation compared to the rest of the formulations tested. It was selected for further development. Stability data of formulations 2414-14 and 2414-15 are summarized in Tables 5 and 6, respectively.

TABLE 4 Wound Healing Gel Formulation Compositions for Stability Testing per Study #1062 Components Formula #: Formula #: Formula #: Formula #: Formula #: Formula #: (% W/W) 2414-10 2414-11 2414-12 2414-13 2414-14 2414-15 API 0.072 0.072 0.072 0.072 0.072 0.072 Propylene glycol 1.0 1.0 1.0 1.0 1.0 1.0 Glycerin 5.0 5.0 5.0 5.0 5.0 5.0 Monobasic Sodium — 0.300 0.263 0.120 0.263 0.263 Phosphate Dibasic Sodium — 0.0036 0.044 0.217 0.044 0.044 Phosphate Methylparaben 0.17 0.17 0.17 0.17 0.17 0.17 Propylparaben 0.02 0.02 0.02 0.02 0.02 0.02 Pluronic 127 20.0 20.0 20.0 20.0 20.0 — (Poloxmer 407) EDTA Disodium — — — — 0.05 0.05 Mannitol — — — — 0.05 0.05 Hydroxyethylcellulose — — — — — 1.25 250HHX Purified water q.s. 100 q.s. 100 q.s. 100 q.s. 100 q.s. 100 q.s. 100 No Buffer Buffer pH 5 Buffer pH 6 Buffer pH 7 Buffer pH 6 Buffer pH 6

TABLE 5 Stability Results of Formula 2414-14 Containing Pluronic 127 per Study #1062 Time Physical Chemical Assay (%, LC) Storage Point Properties and Propyl- Condition (Month) Appearance API Methylparaben paraben Initial Clear and 98.8 92.0 96.6 colorless gel Freeze/Thaw Conforms 89.8 88.6 94.0  5° C. 1 Conforms 84.6 93.0 97.9 25° C. ½ Conforms 90.6 93.6 98.6 1 Conforms 82.8 88.8 94.0

TABLE 6 Stability Results of Formula 2414-15 Containing HEC per Study #1062 Physical Time Properties Chemical Assay (%, LC) Storage Point and Methyl- Condition (Month) Appearance API paraben Propylparaben Initial Clear 101.9 97 103.1 colorless gel Freeze/Thaw Conforms 98.8 97.1 103.9  5° C. 1 Conforms 96 98.1 104.6 3 Conforms 99.7 98.3 101.8 25° C. ½ Conforms 91.6 99.7 106.7 1 Conforms 93.4 96.8 103 3 Conforms 85.7 92.8 96.5

Example 2 R&D Study Per Protocol #1074

Based on the results from study #1062, additional prototype formulations based on formula 2414-15 were designed and evaluated per protocol #1074. During this study, 7 prototype formulations were prepared. The purposes of the study were to select a proper buffering system and antioxidants for the formulation. Five (5) antioxidants were evaluated: Mannitol, Methionine, Acetylcysteine, Lysine and Histidine. Two buffer systems were used: phosphate buffer at pH 6 and citrate buffer at pH 5. Glycerin (5%) was introduced into the new prototype formulations to provide some humectant property to the wound treatment topical gel. PEG 200 was also evaluated for its potential stabilizer effect. The compositions of the prototypes evaluated per study #1074 are listed in Table 7.

TABLE 7 Composition of Prototype Formulations per Study #1074 Formula # 2414- Component (% w/w) 18 19 20 21 22 23 26 Peptide 328967 (ACT1) 0.036 0.036 0.036 0.036 0.036 0.036 0.036 Propylene Glycol 1.0 1.0 1.0 1.0 1.0 — — PEG 200 — — — — — 5.0 5.0 Glycerin 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Monobasic Sodium Phosphate 0.263 0.263 0.263 0.263 0.526 — — Dibasic Sodium Phosphate 0.044 0.044 0.044 0.044 0.088 — — Citric Acid, anhydrous — — — — — 0.60 0.60 Sodium Citrate, anhydrous — — — — — 1.73 1.73 Methylparaben 0.17 0.17 0.17 0.17 0.17 0.17 0.17 Propylparaben 0.02 0.02 0.02 0.02 0.02 0.02 0.02 EDTA disodium 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Mannitol 0.05 — — — — 0.05 — Methionine — 0.5 0.5 0.5 0.5 — — Acetylcysteine — — — — 0.2 — — Lysine — — 0.1 — — — 0.1 Histidine — — — 0.1 — — — Hydroxyethylcellulose 1.25 1.25 1.25 1.25 1.25 1.25 1.25 250 HHX Purified Water, qsad 100 100 100 100 100 100 100 25 mM 25 mM 25 mM 25 mM 50 mM 100 mM 100 mM Buffer Buffer Buffer Buffer Buffer Buffer Buffer pH 6 pH 6 pH 6 pH 6 pH 6 pH 5 pH 5

Due to API availability limitations, 0.036% of API concentration was used for this study. Formula 2414-18 is identical to 2414-15 except that the API concentration was reduced from 0.072% to 0.036%. The available stability data from study #1074 found in Table 8 indicated the following conclusions.

Phosphate buffer seemed to provide better stability to the peptide when compared to the citrate buffer. The buffer capacity at 25 mM is adequate for the system.

Mannitol seemed to provide adequate stability to the peptide.

The effect of other antioxidants was not clear due to the variability in the chemical data.

Based on the study results mainly from study #1062 and partially from #1074, a final formulation was selected for further development. The final formulation contained 25 mM phosphate buffer to maintain the pH of the formulation, mannitol as a stabilizer for the API, and HEC as a gelling agent. The amount of propylene glycol was increased from 1% to 3% in prototype formulations to allow for better solubilization of the parabens in the aqueous system. Four potential API concentrations were selected: 0.0072%, 0.018%, 0.036% and 0.072%. The final formula is summarized in Table 9. The stability of the final selected formula containing different levels of API was also evaluated with analytical methods. In addition, the overall stability was assessed by visual inspection for white powder or precipitate and determination of water content.

TABLE 8 Stability Results of Formulas 2414-18, 2414-19, 2414-20, 2414-21, 2414-22, 2414-23, and 2414-26 from Study #1074 Chemical Assay Time Physical (%, LC) Storage Point Properties Methyl- Propyl- Sample Condition (Month) Appearance API paraben paraben Formula Initial Clear and 106.4 94.6 102.2 2414-18 Colorless Gel 5° C. 1 Conforms 90.1 83.4 88.2 2 Conforms NA NA NA 3 Conforms¹ NA NA NA 25° C. 1 Conforms 94.3 90.4 95.5 2 Conforms NA NA NA 3 Conforms¹ NA NA NA Formula Initial Clear and 109.3 94.2 119.7 2414-19 Colorless Gel 5° C. 1 Conforms 101.1 90.4 112.6 2 Conforms NA NA NA 3 Conforms¹ NA NA NA 25° C. 1 Conforms 103.3 94.6 117.4 2 Conforms NA NA NA 3 Conforms¹ NA NA NA Formula Initial Clear and 107.0 96.9 99.9 2414-20 Colorless Gel 5° C. 1 Conforms 106.8 96.7 98.4 2 Conforms NA NA NA 3 Conforms¹ NA NA NA 25° C. 1 Conforms 97.9 94.2 95.6 2 Conforms NA NA NA 3 Conforms¹ NA NA NA Formula Initial Clear and 104.3 95.1 110.4 2414-21 Colorless Gel 5° C. 1 Conforms 96.7 91.5 104.2 2 Conforms NA NA NA 3 Conforms¹ NA NA NA 25° C. 1 Conforms 96.4 95.0 108.4 2 Conforms NA NA NA 3 Conforms¹ NA NA NA Formula Initial Clear and 112.2 96.1 109.4 2414-22 Colorless Gel 5° C. 1 Conforms 111.4 94.9 106.1 2 Conforms NA NA NA 3 Conforms¹ NA NA NA 25° C. 1 Conforms 96.5 89.5 100.0 2 Conforms NA NA NA 3 Conforms¹ NA NA NA Formula Initial Clear and 87.8 94.7 101.6 2414-23 Colorless Gel 5° C. 1 Conforms 88.3 92.9 97.0 2 Conforms NA NA NA 3 Conforms¹ NA NA NA 25° C. 1 Conforms 74.4 84.4 88.4 2 Conforms NA NA NA 3 Conforms¹ NA NA NA Formula Initial Clear and 83.0 95.5 105.9 2414-26 Colorless Gel 5° C. 1 Conforms 78.5 91.0 98.5 2 Conforms NA NA NA 3 Conforms¹ NA NA NA 25° C. 1 Conforms 77.4 93.3 100.7 2 Conforms NA NA NA 3 Conforms¹ NA NA NA ¹Particles at the bottom of vial; NA = data not available

In one embodiment, a batch of the drug product may be prepared by following the steps of the manufacturing process flow chart and using the composition of Table 9.

Example 3 Treatment of Diabetic Foot Ulcer

A Phase II, randomized, prospective, double blind, parallel group, multi-center study was conducted to assess the safety and efficacy of ACT1 peptide in the treatment of diabetic foot ulcer.

Subjects in Group A received 100 μM of the ACT1 peptide formulation (i.e., Granexin™ Gel) in addition to standard-of-care treatment (SoC) compared to SoC alone in Group B. The total study duration for efficacy assessments was 12 weeks with additional 12 weeks follow up for safety evaluations. The study procedures were divided into three phases: Screening Procedures (Study Days: −7 and 0), Treatment Procedures (Study Day 0 through Study Week 12), and Follow-up Procedures (Study Months 4 through 6).

Subjects enrolled in the study may have had multiple ulcers but only one ulcer was considered as the target ulcer which was within the range of 4-1250 mm² and was separated from other ulcers by at least 20 mm. According to the randomization schedule, subjects had their ulcer site treated with either Granexin™ Gel and SoC or SoC alone. Each subject in Group A was treated with up to 14 applications of Granexin™ Gel over the initial 12 weeks of the study period. Other than the application of Granexin™ Gel, management of the wound sites was equivalent in both the groups. Each subject was assessed weekly for the first 12 weeks of treatment. Statistical analysis of subject data up to 12 weeks was done for efficacy and safety analyses. Wound photographs were used to assess wound closure for both control and test sites. Safety was determined by Treatment Emergent Adverse Events (TEAEs).

Inclusion Criteria:

A subject was enrolled in this study if he/she met the following criteria:

-   1. Male or female aged 18 years and older -   2a. Female subjects were post-menopausal or surgically sterilized,     or -   2b. Female of child-bearing potential must have had a negative     pregnancy test at Screening, and agreed to use hormonal     contraceptive or intra-uterine device or diaphragm with spermicide     or condom with spermicide or abstinence throughout the study -   3. Diabetes mellitus (type I or II) with an HbA1c<10.0% -   4. Diagnosis of neuropathic foot ulcer -   5. Cutaneous, full thickness (University of Texas grade A1), below     ankle surface ulcer between 0.5 cm² and 40 cm² post debridement -   6. A viable, granulating wound as per Investigator's discretion -   7. Ulcer present for at least 4 weeks prior to Screening -   8. An Ankle Brachial Pressure Index (ABPI) between 0.70 and 1.3     measured at Screening -   9. Signed informed consent form

Exclusion Criteria:

-   1. A subject was not eligible for this study if he/she met any of     the following criteria: -   2. Decrease or increase in the ulcer size by 30% or more during 7     day screening period -   3. Cannot tolerate the off-loading method or comply with SoC -   4. An ulcer which showed signs of severe clinical infection, defined     as pus oozing from the ulcer site -   5. The ulcer to be treated required operative debridement -   6. An ulcer positive for β-hemolytic streptococcus upon culture -   7. Requirement for total contact casts -   8. The ulcer had more than 50% slough, significant necrotic tissue,     bone, tendon, or capsule exposure -   9. Highly exuding wounds (wounds that require a daily dressing     change) ABPI<0.7 or >1.3 or ankle systolic pressure<70 mm H -   10. gHad systemic infections -   11. Met one of the following (only 1 out of 3 tests was required): -   a. Doppler waveform analysis on the dorsalis pedis and posterior     tibial arteries a monophasic or biphasic flow (with loss of reverse     flow) in either foot artery, or -   b. A toe: brachial index<0.7 or >1.3, or -   c. Transcutaneous oxygen pressure<40 mm Hg -   12. Presence of an active systemic or local cancer or tumor of any     kind (with the exception of non-melanoma skin cancer) -   13. Congestive Heart Failure New York Heart Association class II-IV     or coronary heart disease with ST segment elevation myocardial     infarction or coronary artery bypass graft or percutaneous     transluminal coronary angioplasty within the last 6 months -   14. Active osteomyelitis of the study foot -   15. Active connective tissue disease -   16. Acute Charcot's neuro-arthropathy as determined by clinical     and/or radiographic examination -   17. Active treatment with systemic corticosteroids -   18. Previous or current radiation therapy to the distal lower     extremity or likelihood to receive this therapy during study     participation -   19. Pregnant or nursing subjects -   20. Uncontrolled anemia (Hb<10 g/dL in females and <12 g/dL in     males) -   21. Estimated glomerular filtration rate<25 mL/min -   22. Poor nutritional status defined as an albumin<25 g/L -   23. Significant peripheral edema as per Investigator's discretion -   24. Known prior inability or unavailability to complete required     study visits during study participation -   25. A psychiatric condition (e.g. suicidal ideation) or chronic     alcohol or drug abuse problem, determined from the subject's medical     history, which, in the opinion of the Investigator, may pose a     threat to subject compliance -   26. Use of a platelet-derived growth factor within 28 days prior to     Screening -   27. Use of any investigational drug or therapy within 28 days prior     to Screening -   28. Any other factor which may have, in the opinion of the     Investigator, compromised participation and follow-up in this study

Granexin™ Gel (100 μM ACT1 peptide, in the formulation described in Table 1 of this application) was applied topically on the wound on Day 0, Day 3, Week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

Total duration of the study was 12 months, including 6 months enrollment period. Individual subject participation for the period of interim analysis was approximately 12 weeks.

Efficacy:

Efficacy was evaluated by assessment of the following parameters between study Day 0 to Week 12:

-   -   Wound photographs*     -   Wound tracings**     -   Wound closure assessment*     -   Wound characteristics (clinical assessment)**     -   Wound treatment response**

Safety:

The safety was evaluated by assessing the following parameters:

-   -   TEAEs (each visit from Day 0 up to Week 12)*     -   Vitals (Day −7 and Week 12 or Last Subject Last Visit [LSLV])**     -   Laboratory assessments (Day −7 and Week 12 or LSLV)** * These         evaluations were considered for the interim analysis** These         evaluations were not considered for the interim analysis

Sample Size Calculations

The sample size was calculated with reference to the primary endpoint (comparing the reduction in wound size as measured by the mean percentage decrease from Baseline to the last visit). Assuming a 25% difference in favor of subjects treated with Granexin™ Gel, power of 80%, significance level of 95% (two-sided) and Standard Deviation (SD) of 40%, 40 subjects were calculated to be required in each treatment group. Adjusting for 15% dropout rate, 92 subjects were required to be enrolled in the study.

All statistical tests were carried out as two-sided on 5% level of significance unless otherwise stated. Summary tables and descriptive statistics were done for demographics, efficacy, and safety variables. To adjust the main efficacy analyses for measured covariates at Baseline, Analysis of Co-Variance (ANCOVA) and survival analysis were performed. The descriptive statistics for continuous variables were presented with number (N) of non-missing observations, mean, SD, median, minimum and maximum or range. For categorical data, descriptive statistics were presented with number of exposed subjects and number (N) with percentage of observations in the various categories of the endpoint, where percentage was based on the exposed subjects.

Primary efficacy analysis was performed on Intent-to-Treat (ITT) and Per Protocol (PP) populations. Primary efficacy data was calculated using sample number (n), mean, SD, median, minimum, maximum, and 95% Confidence Interval (CI).

ANCOVA with repeated measures was performed for comparison of mean percent reduction of wound area from Baseline compared to Week 12 between the two treatment groups. Mean percent reduction of wound area from Baseline to Week 12 was considered as response variable adjusted for the strata, wound duration, viable tissue, exudate level, and the Body Mass Index (BMI) as covariates and treatment group, treatment by visit interaction, and visit as factor included in ANCOVA model.

The mean percent wound closure at 4 weeks compared to Baseline was analyzed as described in the primary efficacy analysis. Subject assessment of intensity of pain at Week 12 was analyzed by the Wilcoxon Mann-Whitney U test.

For time-to-event endpoints, the distribution for each treatment group was estimated by the Kaplan-Meier method and compared by the log-rank test. The median time along with 90% confidence limits was presented for each treatment group. Simultaneous confidence bounds for the Kaplan-Meier curve were computed for both the treatment groups. Further, Cox proportional hazard regression model analysis was conducted to compare the event-time distribution function between the treatment groups. The incidence of 100% study wound closure was a time-to-event analysis computed using Kaplan-Meier methods where all scheduled visits from Day 0 though Week 12 were included in the analysis. This was the primary analysis of the secondary efficacy endpoint. Summary statistics, including the median for incidence of 100% study wound closure was presented. The statistical significance of the difference in the incidence of 100% wound closure was evaluated using the log-rank test. Cox's proportional hazard regression analysis was performed to evaluate the individual and joint effects of several covariates on the incidence of 100% wound closure.

Incidence of 100% wound closure (closed or not closed) by or on Week 12 was analyzed overall and by center. Statistical significance in frequency between treatment groups were assessed by Fisher's Exact test (two-tailed) to determine the overall wound healing frequencies. This was followed by the Cochran-Mantel-Haenszel test which compared the 2 treatment groups with respect to the incidence of 100% wound closure after adjusting for pooled center. The Breslow-Day test was used in conjunction with the Cochran-Mantel-Haenszel test to determine the statistical significance of a treatment by-pooled-center interaction

Results

Mean Percent Wound Closure from Baseline to Week 12

The primary efficacy endpoint of the study was to evaluate mean percent wound closure from Baseline to Week 12. The non-parametric Wilcoxon Mann-Whitney U test was used to analyze mean percent reduction of wound area from Baseline to Week 12 in both the treatment groups since the data did not follow a normal distribution, as confirmed by a Shapiro-Wilk p-value of <0.0001 and a non-linear Q-Q plot. The addition of the ranks of both groups obtained after sorting the data of wound size reduction in ascending order in the PP population showed that the actual sum of scores (1036.00) was higher in Group A subjects than the expected sum of scores i.e. sum of scores when there is no difference in 2 groups, under the null hypothesis (899.00) and in Group B, the actual sum of scores (617.00) was found to be lower than the expected sum of the scores under the null hypothesis (754.00). Therefore, in the PP population, subjects treated with Granexin™ Gel plus SoC (Group A) had a higher percent wound closure from Baseline to Week 12 compared to subjects treated with SoC alone (Group B), which was statistically significantly (p-value=0.0069).

Mean Percent Wound Closure from Baseline to Week 4

Similarly non-parametric analysis for secondary endpoint showed that in the PP population, the mean percent reduction of wound area from Baseline to Week 4 was statistically significantly higher in Group A as compared to Group B (p-value=0.0128).

Subject Self Assessment of Change in Intensity of Pain from Baseline to Week 12

The intensity of pain experienced by the subjects was recorded on a Visual Analogue Scale of 1 to 10 where 1 indicated “no pain” and 10 indicated “extreme pain”. The intensity of pain as assessed by the subjects of the PP population at treatment Week 12 of the study showed using the Wilcoxon Mann-Whitney U test that there was no statistically significant difference in subject self assessment of intensity of pain at Week 12 between the 2 treatment groups (Group A, 0.4±1.22; Group B, 0.3±0.81; p-value=0.9683).

Time-to-Wound Closure Analysis

The time taken for complete wound closure in the PP population was statistically significantly lower in subjects in Group A as compared to subjects in Group B (p-value=0.0057). Of the 32 subjects in Group A, 81.3% subjects had complete (100%) wound closure and 18.8% subjects who did not, were censored. The median duration of 100% wound closure in Group A was 6 weeks. Of the 28 subjects in Group B, 53.6% subjects had complete wound closure and 46.4% subjects not having 100% wound closure, were censored and the median duration of time to complete 100% wound closure was 14.64 weeks.

The time taken to achieve 50% wound closure by Week 12 in the PP population was not statistically significantly lower in subjects in Group A as compared to the time taken by subjects in Group B (p-value=0.2084). The analysis of time taken for complete 50% wound closure by Week 12 showed that of the 32 subjects in Group A, 93.8% subjects completed 50% wound closure and 6.3% subjects did not complete 50% wound closure and were censored. In comparison to that, out of 28 subjects in Group B, 89.3% subjects had 50% wound closure and 10.7% subjects without at least 50% wound closure during the study period were censored. The median duration of 50% wound closure was 2 weeks in Group A and 4.07 weeks in Group B.

Cox Proportional Hazard Regression Analysis for time to 100% wound closure in the PP population showed that though treatment was not a statistically significant factor affecting the wound closure (p-value=0.0856), the subjects in Group A treated with Granexin™ Gel plus SoC would have wound closure 2.339 times more than the subjects in Group B treated with SoC only. Similarly, baseline wound depth and BMI were not statistically significant factors (baseline wound depth: p-value=0.4554, BMI: p-value=0.4435) but would affect wound closure by 1.715 times and 1.050 times, respectively. All other parameters, including wound size, wound duration, and use of dressings did not have a statistically significant effect on the rate of wound closure for both the treatments.

Incidence of 100% Wound Closure by or on Week 12

The categorical analysis of incidence of 100% and 50% wound closure by or on Week 12 in the PP population was done using the Cochran-Mantel-Haenszel analysis with Breslow-Day test and the Chi-square test. The Chi-square test performed by combining the data from all the study centers showed that there were statistically significantly higher number of responders in Group A than in Group B (100% wound closure: p-value=0.0104; 50% wound closure: p-value=0.0063). Therefore it can be concluded that the frequency of 100% and 50% epithelialization occurred at a higher frequency in wounds of subjects treated with Granexin™ Gel than subjects treated with SoC only.

Safety:

Overall, 21 subjects reported 26 AEs of which 18 (69.2%) AEs were mild in intensity. A total of 4 moderate and 4 severe AEs were reported in this study. In Group A, death and foot fracture were the severe AEs and myocardial infarction and inadequate control of diabetes mellitus were severe AEs reported in Group B. None of the AEs reported in this study were considered probably or possibly related to Granexin™ Gel or the SoC treatment. Of the 26 events, 18 (69.2%) recovered and 3 (11.5%) events recovered with sequelae. No action was taken against 22 AEs. None of the subjects withdrew due to an AE. Five subjects reported 5 Serious Adverse Events (SAEs). In Group A, 1 death was reported due to an unknown cause and 1 death in Group B occurred due to myocardial infarction. None of the SAEs, including the deaths, were related to Granexin™ Gel.

This phase II study was a double blind, randomized, prospective, parallel group, multi-center study conducted at 8 centers in India to evaluate the efficacy and safety of Granexin™ Gel in the treatment of DFU. The total duration of the study was 12 months, including 6 months enrollment period and 6 months for study procedures (3 months each for efficacy and safety analyses). Hence, the study duration for an individual subject was approximately 6 months, including 18 visits. For interim analysis, data up to 12 weeks (Visit 15) was considered. A total of 92 subjects were randomized in the study to receive Granexin™ Gel plus SoC or only SoC in the treatment of DFU. Of these subjects, 76 (82.6%) completed the study treatment visits up to Visit 15 and are now in the follow-up stage of the study.

The primary efficacy endpoint of the study was to evaluate mean percent wound reduction from Baseline to Week 12. The non-parametric Wilcoxon Mann-Whitney U test was used to analyze mean percent reduction of wound area from Baseline to Week 12. In the PP population, subjects treated with Granexin™ Gel plus SoC (Group A) showed statistically significantly higher percent wound closure from Baseline to Week 12 compared to subjects treated with SoC alone (Group B) (p-value=0.0069), suggesting that at Week 12 wounds treated with Granexin™ Gel plus SoC healed better than the wounds treated with SoC alone.

The secondary endpoints of the study were to assess mean percent wound closure at Week 4, subject assessment of pain, and time to complete wound closure. At Week 4, Group A subjects in the PP population showed statistically significantly higher percent reduction of wound area from Baseline compared to Group B subjects in the PP population. There was no statistically significant difference in intensity of pain as assessed by the subject from Baseline to Week 12 between the 2 treatment groups. Hence, Granexin™ Gel did not affect the intensity of pain experienced by the subjects as recorded on a Visual Analogue Scale of 1 to 10.

Complete wound closure was defined as 100% epithelialization of the wound and the absence of drainage from the wound. In the PP population, subjects in Group A took significantly less time to achieve 100% wound closure than those in Group B. However, the time taken for 50% wound closure was not significantly lower in Group A as compared to Group B. The categorical analysis of incidence of 100% and 50% wound closure at Week 12 was conducted and the subjects who had 100% wound closure were considered as responders. The number of responders in Group A was significantly higher than in Group B, indicating that the wounds treated with Granexin™ Gel plus SoC have higher incidences of 100% and 50% epithelialization than wounds treated with SoC alone.

Cox Proportional Hazard Regression Model was used to compare the event-time distribution function between the treatment groups. Although treatment was not a significant factor affecting time to 100% wound closure, in the PP population Group A subjects demonstrated 2.3 times higher wound closure than Group B subjects. In addition, baseline wound depth and BMI were not significant factors but affected wound closure by 1.715 times and 1.050 times, respectively. All other parameters, including wound size, wound duration, and use of dressings did not have a statistically significant difference in affecting wound closure for both the treatments. Cox Proportional Hazard Regression Analysis for time to 50% wound closure showed that none of the covariates (treatment group, wound size, wound duration, baseline wound depth, and BMI) were significantly associated with time to 50% wound closure. However, Group A subjects had wound closure 1.186 times more than Group B subjects.

In terms of safety, out of 92 subjects, 21 (22.8%) subjects reported 26 AEs. There was no significant difference in the number of subjects with at least 1 AE between the 2 treatment groups (Group A, 11; Group B, 10; p-value=0.8038). A total of 4 AEs were of severe intensity. None of the AEs were related to the Granexin™ Gel or SoC. None of the subjects withdrew due to an AE. A total 5 (5.4%) subjects reported 5 SAEs where 2 SAEs were observed in Group A subjects and 3 SAEs were in Group B subjects. In Group A, 1 death was reported due to an unknown cause and 1 death in Group B occurred due to myocardial infarction. None of the SAEs were related to Granexin™ Gel or SoC.

In conclusion, Granexin™ Gel along with SoC has shown to accelerate wound healing in subjects with DFU, making it an efficacious, safe, and well tolerated therapeutic option for the treatment of DFU.

The description of 18 visits along with premature study discontinuation and unscheduled visits is summarized below:

Visit 1: Screening (Day −7)

The Screening Visit was held a maximum of 7 days prior to the Baseline Visit. The following procedures were performed at the Screening Visit:

Screening Procedures: informed consent, medical history, physical examination including neuro-vascular examination, vitals, ulcer identification and history, assessment of wound infection, X-rays, Electrocardiogram (ECG), and serum pregnancy test

Inclusion and exclusion criteria checked

Laboratory tests

Immunology tests

Photography of the ulcer

Wound debridement

Wound tracing

Wound bed preparation

Recorded the use of concomitant medication

Recorded the presence of concomitant illness(es)

Visit 2: Baseline (Day 0)

The Baseline Visit was held a maximum of 7 days after Screening. If ulcers healed by >30% within 7 days of the start of the study at Day 0, then the subject was not eligible for participation in the study. The following procedures were performed at Baseline: Inclusion and exclusion criteria checked to confirm that no change had occurred since Screening Randomization Photography of the ulcer Wound tracing Clinical assessment Compliance checked Wound closure assessment Wound bed preparation Treatment with Granexin™ Gel (if applicable) Dressing changed Pain assessment Recorded changes in concomitant medication Recorded Adverse Events (AEs), if any

Visit 3-14: Treatment Visits (Day 3 through Week 11)

On Treatment Visits were conducted at Day 3 followed by Week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 after Baseline Visit±1 day. The following procedures were performed at these visits:

Photography of the ulcer

Wound tracing

Clinical assessment

Compliance checked

Wound closure assessment

Wound bed preparation

Treatment with Granexin™ Gel (if applicable)

Dressing changed

Pain assessment

Recorded changes in concomitant medication

Recorded AEs, if any

Visit 15: End of Treatment Visit (Week 12)

The End of Treatment Visit was conducted at Visit 15 (Week 12±1 day). The following procedures were performed at this visit:

Laboratory tests

Immunology tests

Photography of the ulcer

Wound tracing

Clinical assessment

Compliance checked

Wound closure assessment

Wound bed preparation

Treatment with Granexin™ Gel (if applicable)

Dressing changed

Pain assessment

Recorded changes in concomitant medication

Additional end of treatment visit evaluations (assessment of ulcer response to treatment, comparison of current treatment to previous treatment, incidence of ulcer recurrence within the 12-week treatment period, and vital signs)

Recorded AEs, if any

Visit 16-18: Follow-up Visits, including End of Study Visit (Month 4 through 6) The Follow-up visits would be conducted at Month 4±3 days, Month 5±3 days, and Month 6±3 days (End of Study [EOS] visit). The following procedures would be performed at these visits: Photography of the ulcer Pain assessment Recorded changes in concomitant medication Follow-up procedures (evaluation of ulcer recurrence, care of unhealed ulcers, assessment of healed wounds, and subject assessment of pain) Recorded AEs, if any EOS procedures (only at Visit 18)

Granexin™ Gel is a topical hydrogel that contains the active ingredient ACT1 peptide (100 μM). Granexin™ Gel has greater than 90% water, which provides a barrier to infection or external elements while maintaining a moist environment for the wound. In Group A, the ulcer was cleansed with sterile saline and if any bleeding existed, it was stopped before applying the Granexin™ Gel on Days 0 and 3, and Weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. The Granexin™ Gel was dispensed directly from its 5 ml glass vial(s) to completely cover the wound bed. With a clean sterile cotton swab, sterile tongue depressor, or similar application aid, the Granexin™ Gel was evenly spread over the wound surface. The treatment site was then dressed according to the following regimen:

-   -   Used adequate off-loading other than TCC as per the         Investigator's judgment     -   Applied a non-adherent dressing over the ulcer and Granexin™ Gel         which extended 0.5 inch beyond the ulcer perimeter and inflamed         skin margins     -   Applied a non-occlusive dressing such as fine mesh gauze which         was either folded or rolled as a bolster

Standard of Care (Control) Treatment

In the control group, the ulcer was cleansed with sterile saline and if any bleeding existed, it was stopped before dressing. The ulcer was dressed according to the following regimen:

-   -   Used adequate off-loading other than TCC as per the         Investigator's judgment     -   Applied a non-adherent dressing such as the 4 layer dressing         over the ulcer with either soframycin, Lyseal ointment or         hydroheal AM gel which extended 0.5 inch beyond the ulcer         perimeter and inflamed skin margins     -   Applied a non-occlusive dressing such as pressure bandage or         fine mesh gauze which was either folded or rolled as a bolster

Granexin™ Gel is a topical gel with the active drug substance ACT1 peptide developed at FirstString Research for the management of wounds. ACT1 is a synthetic peptide (25 amino acid) designed to mimic the Carboxy (C)-terminus of the ubiquitous transmembrane gap junction protein Connexin 43 (Cx43), with high binding specificity to zona occludens, a cytoplasmic tight junction protein. While the mechanism of action of ACT1 has not been completely elucidated, the peptide is soluble and engineered to directly translocate within cells. ACT1 peptide interacts with a known binding partner of Cx43—the Postsynaptic Density 95/Discs Large/Zonula Occludens (PDZ)-2 domain of Zonula Occludens-1 (ZO-1). The binding of the peptide to specific Cx43 C-terminus interaction domains such as PDZ-2 on ZO-1 serves to competitively inhibit its association with the Cx43 C-terminus. Connexins are gap junction proteins where as ZO-1 is a tight junction associated protein. The binding of the 25 amino acid ACT1 peptide to the PDZ-2 domain of ZO-1 serves to stabilize gap junctions as well as tight junctions. This junctional stability has been associated with faster healing and reduced scarring. In addition, the low molecular weight (25 amino acid) peptide is expected to reduce the risk of an immunogenic response.

Granexin™ Gel is also designed to provide a localized protective barrier against microbial colonization and a moist environment to promote natural autolytic debridement of necrotic tissue surrounding the wound. The lot number of Granexin™ Gel used in this study was DP 1493.

Subjects were randomized 1:1 to either Group A (Granexin™ Gel plus SoC) or Group B (SoC only). Interactive Web Response System (IWRS) was used for randomization of the eligible subjects to the treatment group. The randomization was further stratified by wound size (<10 cm² and >10 cm²) to avoid bias of undue number of subjects with small or large wounds going into one arm of the study. The subject ID, initials and study Day 0 date were written on the randomization code sheet and filed with the subject's CRF. Randomization lists were prepared centrally by the MNI data management center using a validated computer program (Statistical Analysis Software [SAS] 9.1.3).

Two studies, including a preclinical and clinical study tested the efficacy and safety of 100 μM concentration of ACT1 formulated in Granexin™ Gel. In the preclinical study, the efficacy of Granexin™ Gel in wound healing was assessed on 5 mm diameter full thickness excisional wounds in adult mice and pigs. Wounds treated with 100 μM of ACT1 peptide closed faster, appeared less swollen and inflamed, and healed with a smoother and less discolored appearance as compared to the wounds applied with the vehicle control [18]. Subsequently, a phase I, double blind, single-center, controlled study was conducted to evaluate the safety and tolerability of 20, 50, 100, and 200 μM concentrations of ACT1 formulated in Granexin™ Gel versus placebo in 48 healthy subjects following punch biopsy. The results showed the Granexin™ Gel (ACT1) at each concentration to be safe and well tolerated with no evidence of localized or systemic AEs or Serious Adverse Events (SAEs). Both preclinical and clinical studies concluded 100 μM concentration of the ACT1 peptide to be optimum in safety and effectiveness. Hence, the dose of 100 μM ACT1 of Granexin™ Gel was selected in the treatment of DFU in this study.

For each subject, Granexin™ Gel (100 μM ACT1) was applied at Days 0 and 3, and Weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12

Efficacy and Safety Variables:

The primary efficacy variable was mean percent wound closure from Baseline to Week 12

Secondary Efficacy Variables

-   -   Mean percent wound closure at 4 weeks     -   Subject self-assessment of intensity of pain (till Week 12)     -   Time to 50% wound closure     -   Time to complete wound closure with 100% epithelialization of         the wound

Safety Variable:

The safety variable was incidence of treatment related Adverse Events (AEs) (till Week 12).

An AE was defined as “any untoward medical occurrence in a subject or clinical investigation subject administered a pharmaceutical product that did not necessarily have a causal relationship with this treatment” (ICH E6:1.2). An AE, therefore, was any unfavorable and unintended sign, symptom, or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product.

A clinically significant abnormal laboratory finding was also regarded as an AE and it was the responsibility of the Investigator to review the laboratory test results and determine whether an abnormal laboratory value was of clinical significance. In general, clinically significant laboratory values which suggested disease progression and/or required active management were considered as AEs.

Worsening of a pre-existing medical condition (e.g. diabetes, migraine, headaches, and gout) was considered as an AE if there was either an increase in severity, frequency, duration of the condition, or an association with significantly worse outcomes. Pre-planned procedures and pre-existing conditions that did not worsen were not considered AEs.

A Serious Adverse Event (SAE) was defined as an AE that:

-   -   Was fatal     -   Was life threatening (places the subject at immediate risk of         death)     -   Required in-subject hospitalization or prolongation of existing         hospitalization     -   Resulted in persistent or significant disability/incapacity     -   Was a congenital anomaly/birth defect     -   Met other significant medical hazard

An AE necessitating hospitalization met the regulatory definition for “serious” if the in-subject hospital admission included a minimum of an overnight stay in a health care facility. Any AE that did not meet one of the definitions of serious (e.g. an AE requiring an emergency room visit, out-subject surgery, or requiring urgent investigation) was considered by the Investigator to meet the “other significant medical hazard” criterion for classification as an SAE.

If the above interventions were performed as SoC and not associated with an AE, the health issue for which the intervention was performed was not considered as an SAE. If there was a complication as a result of the procedure and the complication met at least 1 seriousness criterion, then that complication was reported as an SAE.

Study Subjects: A total of 98 subjects with DFU were screened. Of these, 6 were screen failures. The remaining 92 subjects were randomized in the study. Of these subjects, 76 (82.6%) completed the study treatment visits up to Visit 15 and are now in follow-up. The remaining 16 subjects did not complete the study treatment visits up to Visit 15 (Table 9).

TABLE 9 Disposition of Subjects Treatment Group Group A Group B Overall Category (N = 46) (N = 46) (N = 92) Population, n (%)^([1]) Screened 98 Randomized 46 (100.0) 46 (100.0) 92 (100.0) Intent-to-treat 46 (100.0) 45 (97.8) 91 (98.9) Per-Protocol 32 (69.6) 28 (60.9) 60 (65.2) Total number of subjects completed 37 (80.4) 39 (84.8) 76 (82.6) the study (Visit 15) Total number of subjects not  9 (19.6)  7 (15.2) 16 (17.4) completed the study (Visit 15) Note: ^([1])Percentage was calculated taking respective column header group count as denominator.

The following were the reasons, which led to the exclusion of 32 subjects from the PP population: Consent withdrawal (13 subjects); Lost to follow up (2 subjects); Death (2 subjects); Drop outs (1 subject); Protocol deviation(s); Decrease or increase in the ulcer size by 30% or more during 7 day screening period (14 subjects)

ITT Population:

All subjects randomized to any treatment group, received at least one dose of study medication or reference treatment and performed study assessments within and/or outside the time window period specified in the protocol. The assessments performed outside time window period were re-assigned to the closest nominal time point of actual sample collection

PP Analysis Population:

All subjects who completed study assessments within the protocol defined window period for the assigned randomized time point and also completed all study assessments until Week 12. This included subjects who came for all the visits within or outside the window period, including subjects whose wounds had healed before Week 12. Subjects who had a major protocol deviation were excluded from the PP population. This included subjects who had an increase or decrease of more than 30% in wound size during the screening period of 7 days (per exclusion criteria no. 1 in the protocol)

Safety Population:

All subjects who were randomized in the study to either of the treatment groups were used for the safety analysis

The ITT population included 91 subjects; the PP population included 60 subjects; and the safety population included 92 subjects.

Primary Efficacy Endpoint:

Mean Percent Wound Closure from Baseline to Week 12

Parametric Analysis: Table 10 summarizes the parametric analysis of the mean percent reduction of wound area from Baseline to Week 12 in the ITT population. Of 91 subjects in the ITT Population, non-missing observations for change from Baseline to Week 12 for percent reduction of wound area were available for 63 subjects. The mean percent±SD reduction of wound area from Baseline to Week 12 was higher in Group A than Group B (Group A: 72.12%±128.52; Group B: 57.09%±80.88) but was not statistically significant (p-value=0.6567) as per parametric analysis.

TABLE 10 Mean Percent Reduction of Wound Area from Baseline to Week 12 (Parametric analysis) ITT Population (N = 91) Treatment Group Group A Group B Statistics (N = 46) (N = 45) Total number of subjects evaluated N = 63^([1]) Percent reduction of wound area from Baseline to Week 12 n 34 29 Mean 72.121 57.086 SD 128.5222 80.8795 Median 100.000 100.000 Range (Min.:Max.) (−648.91:100.00) (−271.50:100.00) LS Mean Estimate 55.490 44.032 Difference Estimate^([2]) 11.4575 SE^([3]) 25.6968 95% CI (L.:U.) (−39.53:62.44) p-value^([3],[5]) 0.6567 ^([1])“Total no. of subjects evaluated” represents subjects having non-missing observations for change from Baseline to Week 12 for percent reduction of wound area ^([2])Difference estimate for Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plus SoC) - SoC). ^([3])SE of Granexin ™ Gel plus Standard of Care indicated Standard Error of Differences (Granexin ™ Gel plus SoC) - SoC). ^([4])p-value for Granexin ™ Gel plus SoC indicated significance of treatment differences (Granexin ™ Gel plus SoC) - (SoC). ^([5])ANCOVA mixed model with repeated measures was used to compare average percent reduction in wound area with treatment, visit and treatment * visit as factors and strata, wound duration, viable tissue, exuda to level and the BMI as covariates using PROC Mixed procedure of SAS software.

The parametric analysis of the mean percent reduction of wound area from Baseline to Week 12 in the PP population is summarized in Table 11. Of the 60 subjects in the PP population, non-missing observations for change from Baseline to Week 12 for reduction of wound area were available for 57 subjects. The mean percent±SD reduction of wound area from Baseline to Week 12 was statistically significantly higher in Group A than in Group B (Group A: 93.58%±17.67; Group B: 52.14%±84.14; p-value=0.0202).

TABLE 11 Mean Percent Reduction of Wound Area from Baseline to Week 12 (Parametric analysis) PP Population (N = 60) Treatment Group Group A Group B Statistics (N = 32) (N = 28) Total number of subjects evaluated N = 57^([1]) Percent reduction of wound area from Baseline to Week 12 n 31 26 Mean 93.582 52.135 SD 17.6708 84.1427 Median 100.000 100.000 Range (Min.:Max.) (27.61:100.00) (−271.50:100.00) LS Mean Estimate 69.038 30.178 Difference Estimate^([2]) 38.8605 SE^([3]) 16.4998 95% CI (L.:U.) (6.18:71.54) p-value^([4],[5]) 0.0202 Note: ^([1])“Total no. of subjects evaluated” represents subjects having non-missing observations for change from Baseline to Week 12 for percent reduction of wound area ^([2])Difference estimate for Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plus SoC) - SoC). ^([3])SE of Granexin ™ Gel plus Standard of Care indicated Standard Error of Differences (Granexin ™ Gel plus SoC) - SoC). ^([4])p-value for Granexin ™ Gel plus SoC indicated significance of treatment differences (Granexin ™ Gel plus SoC) - (SoC). ^([5])ANCOVA mixed model with repeated measures was used to compare average percent reduction in wound area with treatment, visit and treatment * visit as factors and strata, wound duration, viable tissue, exudate level and the body mass index as covariates using PROC Mixed procedure of SAS software. General Note: Group A indicates Granexin ™ Gel plus SoC and Group B indicates SoC only SD: Standard Deviation, Min: Minimum, Max: Maximum, CI: Confidence Interval, L: Lower Limit, U: Upper Limit Missing data was accounted by using repeated measures analysis. Mean percent reduction from Baseline = ((Baseline − week X)/Baseline) * 100

To confirm the applicability of parametric statistical inference methodology in the wound reduction analysis, it was tested whether the data followed normal distribution. Using PROC UNIVARIATE of SAS® (version 9.1.3) Shapiro-Wilk W statistic was calculated. The Shapiro-Wilk p-value<0.0001 and this indicated that the data did not follow the normal distribution. Therefore, the hypothesis of normal distribution of the data in the sample was rejected and non-parametric analysis was also conducted.

Table 12 summarizes the non-parametric analysis for mean percent reduction of wound area from Baseline to Week 12 in the ITT population. The Wilcoxon Mann-Whitney U test is a non-parametric test which determines whether there is a difference in two samples of independent observations. The actual sum of scores (1215.00) obtained by adding the ranks of both groups after sorting the data of wound size reduction in ascending order, was found higher in Group A subjects than the expected sum of scores i.e. sum of scores when there is no difference in two groups, under the null hypothesis (1088.00) and in Group B, the actual sum of scores (801.00) was found lower than the expected sum of the scores under the null hypothesis (928.00). Subjects in Group A had statistically significantly higher percent reduction of wound area from Baseline to Week 12 than Group B subjects (p-value=0.0287).

TABLE 12 Mean Percent Reduction of Wound Area from Baseline to Week 12 (Non-parametric analysis) IIT Population (N = 91) Treatment Group Group A Group B Statistics (N = 46) (N = 45) Percent reduction of wound area from Baseline to Week 12 Sum of Score 1215.00 801.00 Expected Sum of Scores under H_(o) 1088.00 928.00 p-value^([1]) 0.0287 Note: ^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group. General Note: Mean percent reduction from Baseline = ((Baseline − week X)/Baseline) * 100 H_(o): Null Hypothesis

Table 13 summarizes the non-parametric analysis for mean percent reduction of wound area from Baseline to Week 12 in the PP population. The addition of the ranks of both groups obtained after sorting the data of wound size reduction in ascending order gave the actual sum of scores as 1036.00 which was found to be higher in Group A subjects than the expected sum of scores (899.00) i.e. sum of scores when there is no difference in the two groups, under the null hypothesis and in Group B the actual sum of scores (617.00) was found to be lower than the expected sum of the scores under the null hypothesis (754.00). Subjects in Group A had statistically significant higher percentage of reduction of wound area from Baseline to Week 12 than Group B subjects (p-value=0.0069).

TABLE 13 Mean Percent Reduction of Wound Area from Baseline to Week 12 (Non-parametric analysis) PP Population (N = 60) Treatment Group Group A Group B Statistics (N = 32) (N = 28) Percent reduction of wound area from Baseline to Week 12 Sum of Score 1036.00 617.00 Expected Sum of Scores under H_(o) 899.00 754.00 p-value^([1]) 0.0069 ^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group. General Note: Mean percent Reduction from Baseline = ((Baseline − week X)/Baseline) * 100 H_(o): Null Hypothesis

The parametric evaluation of mean percent wound closure from Baseline to Week 4 in the ITT population is summarized in Table 14. Of 91 subjects in the ITT population, non-missing observations from Baseline to Week 4 for percent reduction of wound area were available for 60 subjects. The mean percent±SD reduction of wound area from Baseline to Week 4 was 53.30%±126.23 in Group A subjects and 52.92%±42.60 in Group B subjects; but the reduction in Group A was not statistically significant compared to Group B (p-value=0.8234).

TABLE 14 Mean Percent Reduction of Wound Area from Baseline to Week 4 (Parametric analysis) ITT Population (N = 91) Treatment Group Group A Group B Statistics (N = 46) (N = 45) Total no. of subjects evaluated N = 60^([1]) Percent reduction of wound area from Baseline to week 4 N 33 27 Mean 53.306 52.920 SD 126.2293 42.6001 Median 81.414 61.484 Range (Min.:Max.) (−626.65:100.00) (−72.94:100.00) LS Mean Estimate 46.931 41.098 Difference Estimate^([2]) 5.8329 SE^([3]) 26.0720 95% CI (L.:U.) (−45.87:57.54) p-value^([4],[5]) 0.8234 Note: ^([1])“Total no. of subjects evaluated” represents subjects having non-missing observations for all the dependent and covariate variables ^([2])Difference estimate for Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plus SoC) - SoC). ^([3])SE of Granexin ™ Gel plus SoC indicated Standard Error of Differences (Granexin ™ Gel plus SoC) - SoC). ^([4])p-value for Granexin ™ Gel plus SoC indicated significance of treatment differences (Granexin ™ Gel plus SoC) - (SoC). ^([5])ANCOVA mixed model with repeated measures was used to compare average percent reduction in wound area with treatment, visit and treatment * visit as factors and strata, wound duration, viable tissue, exudate level and the body mass index as covariates using PROC Mixed procedure of SAS software.

The parametric analysis of mean percent reduction of wound area from Baseline to Week 4 in the PP population is summarized in Table 15. Of the 60 subjects in the PP population, non-missing observations from Baseline to Week 4 were available for a total of 49 subjects. The mean percent±SD reduction of wound area from Baseline to Week 4 was 73.49%±35.52 in Group A subjects and 47.43%±43.61 in Group B subjects; but the reduction was not statistically significantly higher in Group A subjects than Group B (p-value=0.0871).

TABLE 15 Mean Percent Reduction of Wound Area from Baseline to Week 4 (Parametric analysis) PP Population (N = 60) Treatment Group Group A Group B Statistics (N = 32) (N = 28) Total no. of subjects evaluated N = 49^([1]) Percent reduction of wound area from Baseline to week 4 N 26 23 Mean 73.487 47.430 SD 35.5187 43.6110 Median 88.167 56.931 Range (Min.:Max.) (−40.60:100.00) (−72.94:100.00) LS Mean Estimate 53.184 24.019 Difference Estimate^([2]) 29.1649 SE^([3]) 16.9134 95% CI (L.:U.) (−4.31:62.64) p-value^([4], [5]) 0.0871 Note: ^([1])“Total no. of subjects evaluated” represents subjects having non-missing observations for all the dependent and covariate variables ^([2])Difference estimate for Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plus SoC) - SoC). ^([3])SE of Granexin ™ Gel plus SoC indicated Standard Error of Differences (Granexin ™ Gel plus SoC) - SoC). ^([4])p-value for Granexin ™ Gel plus SoC indicated significance of treatment differences (Granexin ™ Gel plus SoC) - (SoC). ^([5])ANCOVA mixed model with repeated measures was used to compare average percent reduction in wound area with treatment, visit and treatment * visit as factors and strata, wound duration, viable tissue, exudate level and the body mass index as covariates using PROC Mixed procedure of SAS software.

Table 16 summarizes the non-parametric analysis for mean percent reduction of wound area from Baseline to Week 4 in the ITT population. In Group A, the expected sum of score under the null hypothesis i.e. sum of scores when there is no difference in two groups was 1006.50 and the actual sum of score was 1148.00 obtained by adding the ranks of both groups after sorting data of wound size reduction in ascending order, and for Group B, it was 823.50 and 682.00 for expected and actual, respectively. Group A subjects had a statistically significantly higher percent reduction of wound area than Group B subjects (p-value=0.0341).

TABLE 16 Mean Percent Reduction of Wound Area from Baseline to Week 4 (Non-parametric analysis) ITT Population (N = 91) Treatment Group Group A Group B Statistics (N = 46) (N = 45) Percent reduction of wound area from Baseline to Week 4 Sum of Score 1148.00 682.00 Expected Sum of Score under Ho 1006.50 823.50 p-value^([1]) 0.0341 Note: ^([1])p-value was evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group. General Note: Mean percent reduction from Baseline = ((baseline − week X)/baseline) * 100 H_(o): Null hypothesis

The non-parametric analysis for mean percent reduction of wound area from Baseline to Week 4 in the PP population is summarized in Table 17 The expected sum of scores, which is the sum of scores when there is no difference in two groups, under the null hypothesis was 650.00 and the actual sum of scores, obtained by adding the ranks of both groups after sorting data of wound size reduction in ascending order, was higher with 773.00 for subjects treated with Granexin™ Gel plus SoC and in subjects treated with SoC only, the expected sum of score was 575.00 and the actual score was lower with 452.00. The percent reduction of wound area in Group A was statistically significantly higher than Group B subjects (p-value=0.0128).

TABLE 17 Mean Percent Reduction of Wound Area from Baseline to Week 4 (Non-parametric analysis) PP Population (N = 60) Treatment Group Group A Group B Statistics (N = 32) (N = 28) Percent reduction of wound area from Baseline to week 4 Sum of Score 773.00 452.00 Expected under H_(o) 650.00 575.00 p-value^([1]) 0.0128 Note: ^([1])p-value was evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group. General Note: Mean percent Reduction from Baseline = ((Baseline − week X)/Baseline) * 100 H_(o): Null Hypothesis

The intensity of pain experienced by the subjects was recorded on a Visual Analogue Scale of 1 to 10 where 1 indicated “no pain” and 10 indicated “extreme pain”. The summary of subject self assessment of intensity of pain at all visits from Baseline to Week 12 for the ITT population have been given in FIG. 2.

Table 18 summarizes the intensity of pain as assessed by the subject at treatment Week 12 of the study. There was no statistically significant difference in the mean intensity of pain from Baseline to Week 12 between the 2 treatment groups (Group A, 0.5±1.46; Group B, 0.3±0.93; p-value=0.8484).

TABLE 18 Analysis of Subject Self Assessment of Intensity of Pain at Week 12 ITT Population (N = 91) Treatment Group Group A Group B Statistics (N = 46) (N = 45) Intensity of pain at Week 12 N 36 38 Mean 0.5 0.3 SD 1.46 0.93 Median 0.0 0.0 Range (Min.:Max.) (0:6) (0:4) Missing 1 0 p-value^([1]) 0.8484 Note: ^([1])p-value was evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group.

Table 19 summarizes the intensity of pain as assessed by the subject at treatment Week 12 of the study for the PP population. There was no statistically significant difference in the mean intensity of pain at Week 12 between the 2 treatment groups (Group A, 0.4±1.22; Group B, 0.3±0.81; p-value=0.9683). The summary of subject self assessment of intensity of pain at all visits from Baseline to Week 12 for the PP population is given in FIG. 3.

TABLE 19 Analysis of Subject Self Assessment of Intensity of Pain at Week 12 PP Population (N = 60) Treatment Group Group A Group B Statistics (N = 32) (N = 28) Intensity of pain at Week 12 n 30 27 Mean 0.4 0.3 SD 1.22 0.81 Median 0.0 0.0 Range (Min.:Max.) (0:4) (0:4) Missing 1 0 p-value^([1]) 0.9683 Note: ^([1])p-value was evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group.

The time taken for 100% wound closure for the ITT population is summarized in FIG. 4. Complete wound closure was defined as 100% epithelialization of the wound and the absence of drainage from the wound. One subject in Group A and 2 subjects in Group B had the visit date missing and were not included in the analysis (Error! Reference source not found. 4). Seventeen (37.0%) subjects in Group A and 25 (55.6%) subjects in Group B were censored. Of these, 2 (4.8%) subjects in Group B were lost-to-follow-up, 1 (2.4%) subject in Group A dropped out, 9 (21.4%) subjects (Group A: 7; Group B: 2) were withdrawn, and 31 (73.8%) subjects (Group A: 10; Group B: 21) did not have complete wound closure during the study period. One patient withdrew from the study but had complete wound closure and was therefore considered in the ITT population, but excluded from the PP population.

In Group A, 28 (60.9%) subjects and 18 (40.0%) subjects in Group B had 100% wound closure. The median duration of 100% wound closure in Group A was 6 weeks (90% CI was 5 to 11 weeks) and 11 weeks (90% CI: 9 to 18.29 weeks) in Group B. In Group B, for 1 subject the assessments were performed outside the window period; the assessment was reassigned to the closest nominal time point which was Visit 15 at 12 weeks. Statistically significantly less time was taken by subjects in Group A to achieve 100% wound closure than Group B subjects (p-value=0.0306).

TABLE 20 Summary of Time to Complete (100%) Wound Closure by Week 12 ITT Population (N = 91) Treatment Group Group A Group B Statistics^([1]) (N = 46) (N = 45) Number (%) of subjects with   28 (60.9%)   18 (40.0%) complete (100%) wound closure Number (%) of censored   17 (37.0%)   25 (55.6%) subjects Median duration (weeks) of 6.00 (5.00; 11.00) 11.00 (9.00; 18.29) complete wound closure (90% CI)^([2]) p-value^([3]) 0.0306 Note: ^([1])Percentage was calculated taking count of corresponding treatment groups as denominator. ^([2])The median duration of complete wound closure was estimated by Kaplan-Meier method and also 90% confidence interval is calculated for the median duration of 100% wound closure. ^([3])p-value is calculated using Log-rank test for treatment groups using Proc Lifetest procedure of SAS software General Note: Time to first complete wound closure where complete wound closure is defined as 100% epithelialization of the wound and the absence of drainage from the wound. Censoring: Subjects without occurrence of complete wound closure during study period, drop outs and lost-to-follow up subjects is censored at the time of last follow-up visit. The data of 1 subject in Group A and 2 subjects in Group B were considered as missing. They had missing data after Screening or had missing visit date.

The time taken to complete (100%) wound closure in the PP population is summarized in Table 21 and FIG. 5 displays the Kaplan-Meier plot for time to 100% wound closure. Of the 32 subjects in Group A, 26 (81.3%) subjects had complete (100%) wound closure and 6 (18.8%) subjects were censored. Subjects who dropped out or were lost to follow-up were already excluded from the PP population and subjects who did not have complete wound closure during the study period were censored. The duration of wound closure was in the range of 4 weeks to 10.86 weeks and the median duration was 6 weeks. Of the 28 subjects in Group B, 15 (53.6%) subjects had complete wound closure and 13 (46.4%) subjects were censored. The duration of wound closure was in the range of 9 weeks to 18.29 weeks. The median duration of time to complete wound closure along with 90% CI was 14.64 weeks. The time taken for complete wound closure was lower in Group A as compared to Group B which was statistically significant (p-value=0.0057).

TABLE 21 Summary of Time to Complete (100%) Wound Closure by Week 12 PP Population (N = 60) Treatment Group Group A Group B Statistics^([1]) (N = 32) (N = 28) Number (%) of subjects with   26 (81.3%)   15 (53.6%) complete (100%) wound closure Number (%) of censored   6 (18.8%)   13 (46.4%) subjects Median duration (weeks) of 6.00 (4.00; 10.86) 14.64 (9.00; 18.29) complete wound closure (90% CI)^([2]) p-value^([3]) 0.0057 Note: ^([1])Percentage was calculated taking count of corresponding treatment groups as denominator ^([2])The median duration of complete wound closure was estimated by Kaplan-Meier method and also 90% confidence interval was calculated for the median duration of 100% wound closure. ^([3])p-value was calculated using Log-rank test for treatment groups using Proc Lifetestt procedure of SAS software

The time taken to complete 50% wound closure by Week 12 in the ITT population is summarized in Table 22 and FIG. 6 displays the Kaplan-Meier plot for time to 50% wound closure in the ITT population. One subject in Group A and 2 subjects in Group B had the visit date missing and were not included in the analysis (FIG. 6). In Group A, 9 (19.6%) subjects and 11 (24.4%) subjects in Group B were censored. Overall, of the 20 censored subjects, 2 (10.0%) subjects were lost-to-follow-up, 1 (5.0%) subject dropped out, 9 (45.0%) subjects were withdrawn, and 8 (40.0%) subjects did not have 50% wound closure during the study period.

Thirty six (78.3%) subjects in Group A and 32 (71.1%) subjects in Group B had 50% wound closure. The median duration of 50% wound closure was 2 weeks (90% CI was 2 to 3.14 weeks) in Group A and 3.29 weeks (90% CI was 3 to 4.14 weeks) in Group B. The time taken for 50% wound closure was not statistically significantly lower in Group A than Group B (p-value=0.5433).

TABLE 22 Summary of Time to Complete 50% Wound Closure by Week 12 ITT Population (N = 91) Treatment Group Group A Group B Statistics^([1]) (N = 46) (N = 45) Number (%) of subjects with   36 (78.3%)   32 (71.1%) 50% wound closure Number (%) of censored   9 (19.6%)   11 (24.4%) subjects Median duration (weeks) of 2.00 (2.00; 3.14) 3.29 (3.00; 4.14) complete wound closure (90% CI)^([2]) p-value^([3]) 0.5433 Note: ^([1])Percentage was calculated taking count of corresponding treatment groups as denominator ^([2])The median duration of complete wound closure was estimated by Kaplan-Meier method and also 90% confidence interval is calculated for the median duration of 50% wound closure. ^([3])p-value was calculated using Log-rank test for treatment groups using Proc Lifetestt procedure of SAS software

The time taken to complete 50% wound closure in the PP population is summarized in Table 23 and FIG. 7 displays the Kaplan-Meier plot for time to 50% wound closure in the PP population. Of the 32 subjects in Group A, 30 (93.8%) subjects completed 50% wound closure and 2 (6.3%) subjects were censored. In Group A, the duration of wound closure was in the range of 2 weeks to 3.14 weeks and the median duration was 2 weeks. Of the 28 subjects in Group B, 25 (89.3%) subjects had 50% wound closure and 3 (10.7%) subjects who did not have at least 50% wound closure during the study period were censored. The duration of wound closure was in the range of 3 weeks to 5 weeks. The median duration of time to complete 50% wound closure was 4.07 weeks. The time taken for complete wound closure was not statistically significantly lower in subjects in Group A as compared to the time taken by subjects in Group B (p-value=0.2084).

TABLE 23 Summary of Time to Complete (50%) Wound Closure by Week 12 PP Population (N = 60) Treatment Group Group A Group B Statistics^([1]) (N = 32) (N = 28) Number (%) of subjects with   30 (93.8%)   25 (89.3%) 50% wound closure Number (%) of censored subjects   2 (6.3%)   3 (10.7%) Median duration (weeks) of 2.00 (2.00; 3.14) 4.07 (3.00; 5.00) complete wound closure (90% CI)^([2]) p-value^([3]) 0.2084 Note: ^([1])Percentage was calculated taking count of corresponding treatment groups as denominator ^([2])The median duration of complete wound closure is estimated by Kaplan-Meier method and also 90% confidence interval is calculated for the median duration of 100% wound closure. ^([3])p-value is calculated using Log-rank test for treatment groups using Proc Lifetestt procedure of SAS software

Additional to the Kaplan-Meier Model, Cox Proportional Hazard Regression Model was used to compare the event-time distribution function between the two treatment groups. This analysis was conducted to evaluate if one or more covariates were associated with the time to complete 100% or 50% wound closure. Table 24 summarizes the Cox Proportional Hazard Regression Analysis of time to complete wound closure in the ITT population. The effect of covariates estimated by the proportional hazard model is reported as hazard ratios. Though treatment was not a significant factor affecting wound closure (p-value=0.2903), the subjects treated with Granexin™ Gel plus SoC would have wound closure 1.568 times more than the subjects treated with SoC only. All other parameters, including wound size, wound duration, baseline wound depth, and use of dressings did not affect wound closure in both the treatment groups.

TABLE 24 Analysis of Time to Complete (100%) Wound Closure ITT Population (N = 91) 95% CI for Hazard Hazard Ratio Statistics^([1]) Ratio^([1]) Lower Upper p-value Treatment group 1.568 0.681 3.607 0.2903 Wound size 0.999 0.997 1.000 0.1265 Wound duration 0.995 0.976 1.013 0.5754 Baseline wound depth 0.782 0.239 2.558 0.6839 BMI 0.966 0.861 1.084 0.5592 Use of dressings — — — — Note: ^([1])Cox Proportional Hazard ratio with 95% confidence interval and p-value was calculated using PROC PHREG procedure.

Cox Proportional Hazard Regression Analysis for time to 100% wound closure in the PP population (Table 25) shows that though treatment was not a significant factor affecting wound closure (p-value=0.0856), the subjects treated with Granexin™ Gel plus SoC would have wound closure 2.339 times more than the subjects treated with SoC only. Similarly, baseline wound depth and BMI were not significant factors (baseline wound depth: p-value=0.4554, BMI: p-value=0.4435) but would affect wound closure by 1.715 times and 1.050 times, respectively. All other parameters, including wound size, wound duration, and use of dressings did not affect wound closure in both the treatment groups.

TABLE 25 Analysis of Time to Complete (100%) Wound Closure (Cox's Proportional Hazards Regression Analysis) PP Population (N = 60) 95% CI for Hazard Hazard Ratio Statistics^([1]) Ratio^([1]) Lower Upper p-value Treatment group 2.339 0.888 6.161 0.0856 Wound size 0.999 0.997 1.000 0.1703 Wound duration 0.992 0.974 1.011 0.4177 Baseline wound depth 1.715 0.416 7.071 0.4554 BMI 1.050 0.927 1.190 0.4435 Use of dressings — — — — Note: ^([1])Cox Proportional Hazard ratio with 95% confidence interval and p-value was calculated using PROC PHREG procedure.

None of the covariates such as treatment group, wound size, wound duration, baseline wound depth, and BMI were significantly associated with time to 50% wound closure. However, in the PP population, subjects treated with Granexin™ Gel plus SoC would have wound closure 1.186 times more than subjects in Group B.

FIG. 8 and FIG. 9 display the average wound closure in the ITT and PP populations in the study, respectively.

Safety evaluation: Brief Summary of Adverse Events

Of 92 subjects, 71 (77.2%) subjects did not report any AEs and 21 (22.8%) subjects reported 26 AEs. Out of 26 AEs, 18 (69.2%) were mild and 2 (15.4%) events in each treatment group were moderate and severe, respectively. A total of 21 (80.8%) AEs were not related and 5 (19.2%) AEs were unlikely related to the study drug or treatment. None of the AEs were related to the study drug. Of the 26 events, 18 (69.2%) recovered and 3 (11.5%) events recovered with sequelae. No action was taken against 22 AEs. None of the subjects withdrew due to an AE.

A total of 5 subjects, of which 2 (4.3%) subjects were in Group A and 3 (6.5%) subjects in Group B, reported 5 SAEs. One subject in Group A died due to an unknown cause and 1 subject in Group B died due to myocardial infarction (Table 26).

TABLE 26 Overview of Adverse Events-Safety Population (N = 92) Treatment Group Group A Group B Overall Statistics (N = 46) (N = 46) (N = 92) Total number of AEs 13 13 26 reported Subjects reporting 11 (23.9%) 10 (21.7%) 21 (22.8%) Any AEs^([1]) Subjects reporting 1 AE 9 (19.6%) 7 (15.2) 16 (17.4%) Subjects reporting >1 AE 2 (4.3%) 3 (6.5%) 5 (5.4%) Subjects reporting 35 (76.1%) 36 (78.3%) 71 (77.2%) No AEs^([1]) Number of AEs with severity of:^([2]) Mild 9 (69.2%) 9 (69.2%) 18 (69.2%) Moderate 2 (15.4%) 2 (15.4%) 4 (15.4%) Severe 2 (15.4%)) 2 (15.4%) 4 (15.4%) Number of AEs with relationship of:^([2]) Not Related 10 (76.9%) 11 (84.6%) 21 (80.8%) Unlikely 3 (23.1%) 2 (15.4%) 5 (19.2%) Possible 0 (0.0%) 0 (0.0%) 0 (0.0%) Probable 0 (0.0%) 0 (0.0%) 0 (0.0%) Definite 0 (0.0%) 0 (0.0%) 0 (0.0%) Number of AEs by outcome:^([2]) Recovered 7 (53.8%) 11 (84.6%) 18 (69.2%) Recovered with sequelae 2 (15.4%) 1 (7.7%) 3 (11.5%) Ongoing when subject 3 (23.1%) 0 (0.0%) 3 (11.5%) completed the study Death 1 (7.7%) 1 (7.7%) 2 (7.7%) Unknown 0 0 0 Number of AEs by action taken:^([2]) None 11 (84.6%) 11 (84.6%) 22 (84.6%) Discontinued study drug 2 (15.4%) 2 (15.4%) 4 (15.4%) Subjects reporting AEs 0 0 0 leading to withdrawal^([1]) Subjects Reporting 2 (4.3%) 3 (6.5%) 5 (5.4%) SAEs^([1]) Subject reporting death 1 (2.2%) 1 (2.2%) 2 (2.2%) Note: ^([1])Percentage was calculated by taking respective column header group count as denominator. ^([2])Percentage was calculated by taking count of ‘Total Number of AEs Reported’ in corresponding treatment group as denominator.

The AEs by MedDRA system organ class and preferred term are summarized in Table 27. There was no statistically significant difference in the number of subjects with at least 1 AE between the 2 treatment groups (p-value=0.8038). In Group A, more than 5% study population reported wound complication (Group A: 5 [10.9%] subjects) and headache (Group A: 4 [8.7%] subjects) as AEs in this study.

TABLE 27 Summary of Adverse Events by MedDRA System Organ Class and Preferred Term Safety Population (N = 92) Treatment Group, n (%)^([1]) Group A Group B Overall System Organ Class/Preferred Term (N = 46) (N = 46) (N = 92) p-value^([2]) Total number of subjects with at 11 (23.9%) 10 (21.7%) 21 (22.8%) 0.8038 least one AEs Total number of AEs reported 13 13 26 Cardiac disorders 0 1 (2.2%) 1 (1.1%) Myocardial infarction^([3]) 0 [0] 1 (2.2%) [1] 1 (1.1%) [1] Gastrointestinal disorders 0 1 (2.2%) 1 (1.1%) Toothache 0 [0] 1 (2.2%) [1] 1 (1.1%) [1] General disorders and 1 (2.2%) 0 1 (1.1%) administration site conditions Death 1 (2.2%) [1] 0 [0] 1 (1.1%) [1] Infections and infestations 0 2 (4.3%) 2 (2.2%) Cellulitis 0 [0] 1 (2.2%) [1] 1 (1.1%) [1] Respiratory tract infection 0 [0] 1 (2.2%) [1] 1 (1.1%) [1] Injury, poisoning and procedural 6 (13.0%) 2 (4.3%) 8 (8.7%) complications Wound complication 5 (10.9%) 2 (4.3%) [3] 7 (7.6%) [9] [6] Foot fracture 1 (2.2%) [1] 0 [0] 1 (1.1%) [1] Metabolism and nutrition disorders 0 1 (2.2%) 1 (1.1) Diabetes mellitus inadequate 0 [0] 1 (2.2%) [1] 1 (1.1%) [1] control Musculoskeletal and connective 0 (6.5%) 3 (3.3%) tissue disorders Back pain 0 [0] 1 (2.2%) [1] 1 (1.1%) [1] Pain in extremity 0 [0] 2 (4.3%) [2] 2 (2.2%) [2] Nervous system disorders 4 (8.7%) 1 (2.2%) 5 (5.4%) Headache 4 (8.7%) [4] 1 (2.2%) [1] 5 (5.4%) [5] Respiratory, thoracic and 1 (2.2%) 0 1 (1.1%) mediastinal disorders Pneumonitis 1 (2.2%) [1] 0 [0] 1 (1.1%) [1] Skin and subcutaneous tissue 0 1 (2.2) 1 (1.1%) disorders Blister 0 [0] 1 (2.2%) [1] 1 (1.1%) [1] Note: ^([1])Percentage was calculated by taking respective column header group count as denominator. ^([2])p-value was calculated by comparing two treatment group using Chi-square test. ^([3])Myocardial infarction led to death of the subject

Out of 26 AEs, 2 events each in both the treatment groups were considered severe AEs. In both Group A and B, 9 (69.2%) AEs each were mild, 2 (15.4%) AEs each were moderate and 2 (15.4%) AEs each were severe. The AEs with severe intensity were death and foot fracture in Group A and myocardial infarction and inadequate control of diabetes mellitus in Group B. None of the severe AEs were related to the study drug or study treatment

Overall, 10 (76.9%) AEs in Group A and 11 (84.6%) AEs in Group B were not related, 3 (23.1%) AEs in Group A and 2 (15.4%) AEs in Group B were unlikely related. None of the AEs were related to Granexin™ Gel or study treatment.

Two AEs of death (Subject 903) and foot fracture (Subject 504) in Group A led to study drug discontinuation. In Group B, two events of cellulitis (Subject 524) and wound complication (Subject 806 with increase in size of ulcer) led to discontinuation of study treatment.

Three events recovered with sequelae (Group A, foot fracture [Subject 504] and wound complication [Subject 508]; Group B, right foot cellulitis [Subject 524]). One subject in each treatment group reported death (Group A, unknown cause [Subject 903]; Group B, myocardial infarction [Subject 827]).

Table 28 summarizes SAEs by MedDRA system organ class and preferred term. Five (5.4%) subjects (Group A: 2 subjects; Group B: 3 subjects) reported SAEs where foot fracture (Subject 504) and death due to unknown cause (Subject 903) were reported by Group A subjects; and myocardial infarction (Subject 827), diabetes mellitus inadequate control (Subject 201), and cellulitis (Subject 524) were reported by Group B subjects.

TABLE 28 Summary of Serious Adverse Events by MedDRA System Organ Class and Preferred Term Safety Population (N = 92) Treatment Group, n (%)^([1]) Group A Group B Overall System Organ Class/Preferred Term (N = 46) (N = 46) (N = 92) Total number of subjects with at 2 (4.3) 3 (6.5) 5 (5.4) least one SAE Total number of SAEs 2 3 5 Cardiac disorders 0 (0.0) 1 (2.2) 1 (1.1) Myocardial infarction^([2]) 0 (0.0) [0] 1 (2.2) [1] 1 (1.1) [1] General disorders and administration 1 (2.2) 0 (0.0) 1 (1.1) site conditions Death 1 (2.2) [1] 0 (0.0) [0] 1 (1.1) [1] Infections and infestations 0 (0.0) 1 (2.2) 1 (1.1) Cellulitis 0 (0.0) [0] 1 (2.2) [1] 1 (1.1) [1] Injury, poisoning and procedural 1 (2.2) 0 (0.0) 1 (1.1) complications Foot fracture 1 (2.2) [1] 0 (0.0) [0] 1 (1.1) [1] Metabolism and nutrition disorders 0 (0.0) 1 (2.2) 1 (1.1) Diabetes mellitus inadequate control 0 (0.0) [0] 1 (2.2) [1] 1 (1.1) [1] Note: ^([1])Percentage was calculated by taking respective column header group count as denominator. ^([2])Myocardial infarction led to death of the subject.

The 5 SAEs with the outcome are summarized in Table 29 below:

TABLE 29 SAEs by Severity, Outcome, and Action Taken Subject Relationship to Study ID Severity Drug/Study Treatment Action Taken Outcome 903 Severe Not related None Fatal 827 Severe Not related None Fatal 201 Severe Not related None Recovered 504 Severe Not related Discontinued Recovered Study drug with sequelae 524 Moderate Not related Discontinued Recovered Study drug with sequelae

Safety Conclusions: Overall, 21 subjects reported 26 AEs of which majority were mild in intensity. A total of 4 moderate and 4 severe AEs were reported in this study. In Group A, death and foot fracture were the severe AEs and myocardial infarction and inadequate control of diabetes mellitus were severe AEs reported in Group B. All the severe AEs were not related to study treatment. None of the AEs reported were related to Granexin™ Gel or study treatment. Of the 26 events, 18 (69.2%) recovered and 3 (11.5%) events recovered with sequelae. No action was taken against 22 AEs. Study drug or treatment was discontinued in a 2 subjects each in Group A and Group B. None of the subjects withdrew due to an AE. Five subjects reported 5 SAEs. None of the SAEs, including 1 fatal event in Group A, were related to Granexin™ Gel.

Discussion and Overall Conclusions:

This was a double blind, randomized, prospective, parallel group, multi-center Phase II study conducted at 8 centers in India to evaluate the efficacy and safety of Granexin™ Gel in the treatment of DFU. The total duration of the study was 12 months, including 6 months enrollment period and 6 months for study procedures (3 months each for efficacy and safety analyses). Hence, the study duration for an individual subject was approximately 6 months, including 18 visits. For interim analysis, data up to 12 weeks (Visit 15) was considered. A total of 98 subjects with DFU were screened for this study and 6 were found to be screen failures. The remaining 92 subjects were randomized in the study to receive Granexin™ Gel plus SoC or only SoC in the treatment of DFU. Of these subjects, 76 (82.6%) completed the study treatment visits up to Visit 15 and are now in the follow-up stage of the study.

The primary efficacy endpoint of the study was to evaluate mean percent wound closure from Baseline to Week 12. The non-parametric Wilcoxon Mann-Whitney U test was used to analyze mean percent reduction of wound area from Baseline to Week 12 in both the treatment groups since the data did not follow a normal distribution, as confirmed by a Shapiro-Wilk p-value<0.0001 and a non-linear Q-Q plot. In the PP population, subjects treated with Granexin™ Gel plus SoC (Group A) showed statistically significantly higher percent wound closure from Baseline to Week 12 compared to subjects treated with SoC alone (Group B) (p value=0.0069), suggesting that at Week 12 wounds treated with Granexin™ Gel plus SoC healed better than the wounds treated with SoC alone.

The secondary endpoints of the study were to assess mean percent wound closure at Week 4, subject assessment of pain, and time to complete wound closure. At Week 4 in the PP population, Group A subjects showed statistically significantly higher percent reduction of wound area from Baseline compared to Group B subjects (p-value=0.0128; Wilcoxon Mann-Whitney U test). There was no statistically significant difference in intensity of pain as assessed by the subject from Baseline to Week 12 between the 2 treatment groups (p-value=0.8484; Wilcoxon Mann-Whitney U test), hence Granexin™ Gel did not affect the intensity of pain experienced by the subjects as recorded on a Visual Analogue Scale of 1 to 10.

Complete wound closure was defined as 100% epithelialization of the wound and the absence of drainage from the wound. Group A subjects in the PP population took significantly less time to achieve 100% wound closure than Group B subjects in the PP population (median duration: Group A, 6 weeks; Group B, 14.64 weeks; p-value=0.0057; log-rank test). However, in the PP population the time taken for 50% wound closure was not significantly lower in Group A as compared to Group B (median duration: Group A, 2 weeks; Group B, 4.07 weeks; p-value=0.2084; log-rank test). The categorical analysis of incidence of 100% and 50% wound closure at Week 12 was conducted and the subjects who had 100% wound closure were considered as responders. The number of responders in Group A in the PP population was significantly higher than in Group B (100% wound closure: p-value=0.0104; 50% wound closure: p-value=0.0063; Chi-square analysis), indicating that the wounds treated with Granexin™ Gel plus SoC have a higher frequency of 100% and 50% epithelialization than wounds treated with SoC alone.

Cox Proportional Hazard Regression Model was used to compare the event-time distribution function between the treatment groups. This analysis was conducted to evaluate if one or more covariates were associated with the time to complete 100% or 50% wound closure. Cox Proportional Hazard Regression Analysis for time to 100% wound closure showed that in the PP population though treatment was not a significant factor affecting wound closure. Group A subjects demonstrated 2.3 times higher wound closure than Group B subjects. In addition, baseline wound depth and BMI were not significant factors but affected wound closure by 1.715 times and 1.050 times, respectively. All other parameters, including wound size, wound duration, and use of dressings did not have a statistically significant difference in affecting wound closure for both the treatments. Cox Proportional Hazard Regression Analysis for time to 50% wound closure showed that none of the covariates (treatment group, wound size, wound duration, baseline wound depth, and BMI) were significantly associated with time to 50% wound closure. However, Group A subjects had 1.186 times more wound closure than Group B subjects.

The safety variable was to evaluate the incidence of treatment related AEs. Out of 92 subjects, 21 (22.8%) subjects reported 26 AEs. There was no significant difference in the number of subjects with at least 1 AE between the 2 treatment groups (Group A, 11; Group B, 10; p-value=0.8038). A total of 4 AEs were of severe intensity. None of the AEs were related to the Granexin™ Gel. None of the subjects withdrew due to an AE. A total of 5 (5.4%) subjects reported 5 SAEs of which 2 were Group A subjects and 3 were Group B subjects, none of the SAEs was related to Granexin™ Gel or study treatment. In Group A, 1 death was reported due to an unknown cause and 1 death in Group B occurred due to myocardial infarction.

In conclusion, Granexin™ Gel along with SoC has shown to accelerate wound healing in subjects with DFU, making it an efficacious, safe, and well tolerated therapeutic option for the treatment of DFU.

Example 4 Treatment of Venous Leg Ulcers

A Phase II, Randomized, Prospective, Double Blind, Parallel Group, Multi-Center Study to Determine the Safety and Efficacy of Granexin™ Gel in the Treatment of Venous Leg Ul

This was a double blind, multi-center, phase II study to evaluate the efficacy and safety of Granexin™ Gel in the treatment of Venous Leg Ulcer (VLU). Total duration of the study was 11 months. The enrollment period of the study was 5 months and 6 months for study procedures. Study duration for individual subjects was 6 months and each subject was to attend a total of 18 visits during the study. Subjects were randomized to 1 of 2 treatment groups: Group A: Granexin™ Gel plus Standard Of Care (SoC) in treatment of VLU Group B: SoC in treatment of VLU Subjects in Group A received Granexin™ Gel (100 μM ACT1 peptide) plus SoC compared to SoC alone in Group B. The total study duration for efficacy assessments was 12 weeks with additional 12 weeks follow up for safety evaluations. The study procedures were divided into 3 phases: Screening Phase (Day −7 through Day 0), Treatment Phase (Day 0 through Week 12), and Follow-up Phase (Month 4 through Month 6).

Subjects enrolled in the study may have had multiple ulcers but only one ulcer was considered as the target ulcer. According to the randomization schedule, subjects had their ulcer site treated with either Granexin™ Gel and SoC or SoC alone. Each subject in Group A was treated up to 14 applications of Granexin™ Gel over the initial 12 weeks of the study period. Other than the application of Granexin™ Gel for Group A subjects, management of the wound sites was similar in both groups.

Each subject was assessed weekly for the first 12 weeks, followed by monthly assessments for the next 3 months. Wound photographs were used to assess wound closure in all subjects. Safety was determined by Treatment Emergent Adverse Events (TEAEs).

The primary end point of the study is:

-   -   The mean percent wound closure from Baseline to 12 weeks

The secondary end points are:

-   -   Mean percent wound closure at 4 weeks     -   Time to 50% wound closure     -   Time to complete wound closure where complete wound closure will         be defined as 100% epithelialization of the wound and the         absence of drainage from the wound     -   Subject self-assessment of intensity of pain     -   Incidence of treatment related adverse events (AE)

Inclusion Criteria:

A subject was enrolled in this study if he/she met the following criteria:

-   2. Male or female aged 18 years and older -   2a. Female subjects were post-menopausal or surgically sterilized,     or -   2b. Female of child-bearing potential must have had a negative     pregnancy test at Screening, and agreed to use hormonal     contraceptive or intra-uterine device or diaphragm with spermicide     or condom with spermicide or abstinence throughout the study -   3. Subjects with ulcers of venous origin, as clinically determined     by the Investigator by a positive venous reflux test (venous     refilling<20 seconds) using Doppler Ultrasound for at least 4 weeks     prior to screening day, which have not adequately responded to     conventional ulcer therapy -   4. Ulcers that extend through the epidermis but not through muscle,     tendon, or bone (Stage II or III ulcers as defined by the     International Association of Enterostomal Therapists) -   5. Surface ulcer between 0.5 cm² and 40 cm² post debridement -   6. Signed informed consent form

Exclusion Criteria:

A subject was not eligible for this study if he/she met any of the following criteria:

-   1. Decrease or increase in the ulcer size by 30% or more during 7     day screening period -   2. Cannot tolerate or comply with compression therapy -   3. An ulcer which showed signs of severe clinical infection, defined     as pus oozing from ulcer site -   4. The ulcer to be treated required operative debridement -   5. An ulcer positive for β-hemolytic Streptococcus upon culture -   6. The ulcer had more than 50% slough, significant necrotic tissue,     osteomyelitis, bone, tendon, or capsule exposure or avascular ulcer     beds -   7. Highly exuding wounds (wounds that required a daily dressing     change) df -   8. Ankle Brachial Pressure Index (ABPI)<0.65 -   9. With active systemic infections -   10. With clinically significant medical conditions as determined by     the Investigator which would impair wound healing including renal,     hepatic, hematologic, neurologic, or immune disease. Examples     included but were not limited to     -   a. Renal insufficiency as an estimated glomerular filteration         rate which was <30 mL/min/1.7 m²     -   b. Abnormal blood biochemistry defined as 3 times that of the         upper limit of the normal range     -   c. Hepatic insufficiency defined as total bilirubin>2 mg/dL or         serum albumin<25 g/L     -   d. HbA1c>9.0%     -   e. Hemoglobin<10 g/dL     -   f. Hematocrit<0.30     -   g. Platelet count<100,000 -   11. Presence of an active systemic or local cancer or tumor of any     kind (with the exception of non-melanoma skin cancer) -   12. With active osteomyelitis of the study foot -   13. With severe rheumatoid arthritis (with more than 20 persistently     inflamed joints, or below lower normal limit blood albumin level, or     evidence of bone and cartilage damage on X-ray, or inflammation in     tissues other than joints) and other collagen vascular diseases -   14. With active connective tissue disease -   15. Treatment with systemic corticosteroids (>15 mg/day), or current     immunosuppressive agents -   16. Previous or current radiation therapy or likelihood to receive     this therapy during study participation -   17. Pregnant or nursing subjects -   18. Known prior inability or unavailability to complete required     study visits during study participation -   19. A psychiatric condition (e.g. suicidal ideation) or chronic     alcohol or drug abuse problem, determined from the subject's medical     history, which, in the opinion of the Investigator, may pose a     threat to subject compliance -   20. Use of any investigational drug or therapy within the 28 days     prior to Screening -   21. Any other factor which may have, in the opinion of the     Investigator, compromised participation and follow-up in this study

Granexin™ Gel (100 μM ACT1 peptide) was applied topically on the wound on Day 0 (Baseline visit), Day 3, Week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. The duration of the study was 11 months which included 5 months enrollment period. Individual subject participation was approximately for the duration of 6 months.

The reference therapy used in this study was SoC for VLU. The ulcer was cleaned with sterile saline and bleeding was stopped before dressing. The ulcer was dressed according to the following regimen:

-   -   Applied a non-adherent dressing over the ulcer which extended         0.5 inch beyond the ulcer perimeter and inflamed skin margins     -   Applied a non-occlusive dressing such as fine mesh gauze which         was either folded or rolled as a bolster     -   Applied a self adherent high compression four layer elastic wrap         (3M Elastic Adhesive Bandage) or a compression crepe bandage         from metatarsals to tibial plateau so that therapeutic         compression is applied to the ulcer site

This was a double blind, randomized, prospective, parallel group, multi-center Phase II study conducted at 10 centers in India to evaluate the efficacy and safety of Granexin™ Gel in the treatment of VLU. The total duration of the study was 11 months, including 5 months enrollment period and 6 months for study procedures. The study duration for individual subjects was 6 months which included 18 visits. A total of 101 subjects with VLU were screened for this study, 5 were found to be screen failures, and 4 subjects did not initiate participation in the study. A total of 92 subjects enrolled in the study had mean wound duration of 68.4 weeks at Screening and mean wound area of 353.64 mm² at Baseline. These subjects were randomized to receive Granexin™ Gel plus SoC or SoC alone for the treatment of VLU. Of these, 70 completed the study and 22 subjects did not complete the study due to not meeting exclusion criterion number 1, consent withdrawal, loss to follow-up, non-compliance, or SAE. Of 92 enrolled subjects, 15 subjects met exclusion criterion 1 but were considered in the Safety and ITT population. These subjects were excluded from the mITT and PP populations. A total of 83 (90.2%) subjects were males and 9 (9.8%) were females. The mean age of the study population was 49.8 years, mean weight was 70.47 kg, mean height was 168.35 cms, and mean BMI was 24.79 kg/m².

This was a phase II, prospective, randomized, double blind, parallel group, and multi-center study conducted in India in subjects with VLU. The total study duration was 11 months. The enrollment period was 5 months and 6 months for study procedures. Study duration for individual subjects was 6 months and each subject attended a total of 18 visits, including Screening (Visit 1), Baseline, (Visit 2), Treatment (Visit 3-15), and Follow-up (Visit 16-18) visits. The study procedures were divided into 3 phases: Screening Phase (Days −7 through Day 0), Treatment Phase (Day 0 through Week 12), and Follow-up Phase (Months 4 through Month 6). Subjects were randomized to 1 of the 2 treatment groups:

-   -   Group A: Granexin™ Gel plus SoC     -   Group B: SoC only

Only one ulcer per subject was considered as the target ulcer which was the largest ulcer. Each subject in the Granexin™ Gel treatment group was treated with up to 14 applications of Granexin™ Gel over the initial 12 weeks of the study period. Other than the application of Granexin™ Gel, management of ulcer was similar in both the groups.

The primary efficacy endpoint of the study was to evaluate mean percent wound closure from Baseline (Visit 2) to Week 12 (Visit 15). The non-parametric Wilcoxon Mann-Whitney U test was used to analyze mean percent reduction of wound area from Baseline to Week 12 in both the treatment groups since the data did not follow a normal distribution, as confirmed by a Shapiro-Wilk p-value<0.0001 and a non-linear Q-Q plot. Subjects treated with Granexin™ Gel plus SoC showed statistically significantly higher percent wound closure from Baseline to Week 12 compared to subjects treated with SoC alone (ITT p-value=0.0238; mITT p-value=0.0105; PP p-value=0.0073), suggesting that at Week 12, wounds treated with Granexin™ Gel plus SoC reduced in size when compared to wounds treated with SoC alone.

Mean percent wound closure at Week 4, time to complete (100% and 50%) wound closure, incidence of complete wound closure, and subject self assessment of pain were also analyzed. At Week 4, a higher percentage reduction of wound area was observed in Granexin™ Gel plus SoC group than in the SoC group and this was statistically significant (ITT p-value=0.0006; mITT p-value=0.0001; PP p-value<0.0001 Wilcoxon Mann-Whitney U test).

Subjects treated with Granexin™ Gel plus SoC achieved 100% wound closure in median duration of 6 weeks in ITT, mITT, and PP population which was significantly faster compared to the SoC group in which the median duration was 12.14 weeks in the ITT population and not achieved in both mITT and PP population during the 12 weeks of efficacy assessments (ITT p-value=0.0006; mITT p-value=0.0001; PP p-value<0.0001; log-rank test).

Subjects treated with Granexin™ Gel plus SoC achieved 50% wound closure in 2.86 weeks in ITT, mITT and PP population while subjects treated with SoC alone achieved it in 6.86 weeks in ITT population and 8 weeks in both mITT and PP population (ITT p-value=0.0002, mITT p-value<0.0001, PP p-value<0.0001). The categorical analysis of incidence of 100% and 50% wound closure at Week 12 was conducted and the subjects who had 100% and 50% wound closure were considered as responders. Overall in the ITT, mITT, and PP populations, 56.5%, 63.4%, and 74.3% of the subjects in Granexin™ Gel plus SoC group, respectively, responded to treatment with 100% wound closure as compared to 30.3% of the subjects in the SoC group. The number of responders were significantly higher in Granexin™ Gel plus SoC group than in the SoC group (ITT: p-value=0.0061; mITT p-value=0.0040; PP: p-value=0.0003; Chi-square analysis). Similarly, in the PP population, 80.0% of the subjects in Granexin™ Gel plus SoC group responded to treatment and achieved 50% wound closure as compared to 51.5% of the subjects in the SoC group. The number of responders with 50% wound closure were significantly higher in Granexin™ Gel plus SoC group than in the SoC group (p-value=0.0131; Chi-square analysis), indicating that the wounds treated with Granexin™ Gel plus SoC have higher incidences of 100% and 50% wound closure (epithelialization) than wounds treated with SoC alone.

Cox Proportional Hazard Regression Model analysis was conducted to evaluate if one or more covariates such as treatment group, wound duration, baseline wound depth, and BMI etc. were associated with the time to complete 100% or 50% wound closure. It showed that treatment group was a significant factor affecting wound closure and Granexin™ Gel plus SoC group subjects had ITT: 2.281; mITT: 2.776; PP: 3.150 times more chances of 100% wound closure and ITT: 2.247; mITT: 2.501; PP: 2.61 times more chances of 50% wound closure than the SoC group subjects.

At every visit, self assessment of intensity of pain score was completed by each subject and the scores were given in comparison to the last visit. Therefore significant change in the pain scores was not observed and there was no statistically significant difference in intensity of pain as assessed by the subject at Week 12 between the 2 treatment groups.

The findings from the present study demonstrated a good safety profile with Granexin™ Gel. Overall, 28 subjects reported 40 AEs of which 24 AEs were reported by subjects in Granexin™ Gel plus SoC group; 16 AEs were reported in the SOC group. The only 2 SAEs reported, were in the SoC group. One SAE was infarction which resulted in death, and the other SAE was Deep Vein Thrombosis. Of the 40 AEs, 35 (87.5%) recovered. None of the AEs were related to Granexin™ Gel. None of the subjects were withdrawn from the study due to an AE.

No clinically significant abnormalities in the laboratory parameters were observed in either of the treatment groups. The vital signs and ECG were normal and comparable between both the treatment groups. Anti-ACT1 peptide antibodies were not detected in the serum samples collected at Screening or at Week 12.

In conclusion, Granexin™ Gel along with SoC has shown to accelerate wound healing in subjects with VLU, making it an efficacious, safe, and well tolerated therapeutic option in the treatment of VLU.

Description of Study Visits: The description of 18 visits along with premature study discontinuation and unscheduled visits is summarized below:

Visit 1: Screening Visit (Day −7): The Screening visit was held a maximum of 7 days prior to the Baseline visit.

Visit 2: Baseline Visit (Day 0)

The Baseline visit was conducted within 7 days of the Screening visit. If wound size increased or decreased by 30% on the day of randomization from Screening (exclusion criterion number 1), then the subject was considered ineligible for participation in the study. The following procedures were performed at the Baseline visit:

Visit 3-14: Treatment Visits (Day 3 through Week 11)

The treatment visits were conducted at Day 3 and at Week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 after Baseline visit±1 day. The following procedures were performed at these treatment visits:

Visit 15: End of Treatment Visit (Week 12)

Visit 16-18: Follow-up Visits including End of Study Visit (Month 4 through 6):

The Follow-up visits were conducted at Month 4±3 days, Month 5±3 days, and Month 6±3 days (End of Study [EOS] visit). The following procedures were performed at this visit:

Standard of Care Treatment

The SoC was followed in both treatment groups. The ulcer was cleaned with sterile saline and bleeding was stopped before dressing. The ulcer was dressed according to the following regimen:

-   -   Applied a non-adherent dressing over the ulcer which extended         0.5 inch beyond the ulcer perimeter and inflamed skin margins     -   Applied a non-occlusive dressing such as fine mesh gauze which         was either folded or rolled as a bolster     -   Applied a self adherent high compression four layer elastic wrap         (3M Elastic Adhesive Bandage) or a compression crepe bandage         from metatarsals to tibial plateau so that therapeutic         compression is applied to the ulcer site

In the Granexin™ Gel plus SoC group, the SoC as mentioned above was followed along with the application of Granexin™ Gel. The ulcer was cleaned with sterile saline and bleeding was stopped before applying the Granexin™ Gel on Days 0 and 3, and Weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. The Granexin™ Gel was dispensed directly from its 5 ml glass vial(s) to completely cover the wound bed. With a clean sterile cotton swab, sterile tongue depressor, or a similar application aid, the Granexin™ Gel was evenly spread over the wound surface.

Granexin™ Gel is a topical gel with the active drug substance Alpha-Connexin C-terminal peptide 1 (ACT1) peptide developed at FirstString Research for the management of wounds. ACT1 is a synthetic peptide (25 amino acid) designed to mimic the Carboxy (C)-terminus of the ubiquitous transmembrane gap junction protein Connexin 43 (Cx43), with high binding specificity to zona occludens, a cytoplasmic tight junction protein. While the mechanism of action of ACT1 has not been completely elucidated, the peptide is soluble and engineered to directly translocate within cells. ACT1 peptide interacts with a known binding partner of Cx43—the Postsynaptic Density 95/Discs Large/Zonula Occludens (PDZ)-2 domain of Zonula Occludens-1 (ZO-1). The binding of the peptide to specific Cx43 C-terminus interaction domains such as PDZ-2 on ZO-1 serves to competitively inhibit its association with the Cx43 C-terminus. Connexins are gap junction proteins where as ZO-1 is a tight junction associated protein. The binding of the 25 amino acid ACT1 peptide to the PDZ-2 domain of ZO-1 serves to stabilize gap junctions as well as tight junctions [12]. This junctional stability has been associated with faster healing and reduced scarring. In addition, the low molecular weight of 25 amino acid peptide is expected to reduce the risk of an immunogenic response.

Granexin™ Gel is also designed to provide a localized protective barrier against microbial colonization and a moist environment to promote natural autolytic debridement of necrotic tissue surrounding the wound. The lot number of Granexin™ Gel used in this study was DP 1493.

Central block randomization (Block size of 2) list was prepared by MNI independent Biostatistician using a validated computer program (Statistical Analysis Software [SAS] 9.1.3). Subjects were randomized 1:1 to either Group A (Granexin™ Gel plus SoC) or Group B (SoC only). Randomization was further stratified by wound size (<10 cm² and >10 cm²) to avoid bias of undue number of subjects with small or large wounds going into one arm of the study.

Interactive web response system (IWRS) was used for treatment group assignment of the eligible subjects. Being the double-blinded study, an unblinded cordinator designated by the Investigator received the assigned treatment through IWRS.

Two studies, including a preclinical and clinical study tested the efficacy and safety of 100 μM concentration of ACT1 formulated in Granexin™ Gel. In the preclinical study, the efficacy of Granexin™ Gel in wound healing was assessed on 5 mm diameter full thickness excisional wounds in adult mice and pigs. Wounds treated with 100 μM of ACT1 peptide closed faster, appeared less swollen, inflamed and healed with a smoother and less discolored appearance as compared to the wounds applied with the vehicle control. Subsequently, a Phase I, double blind, single-center, controlled study was conducted to evaluate the safety and tolerability of 20, 50, 100, and 200 μM concentrations of ACT1 formulated in Granexin™ Gel versus placebo in 48 healthy subjects following punch biopsy. The results showed Granexin™ Gel (ACT1) at each concentration to be safe and well tolerated with no evidence of localized or systemic AEs or Serious Adverse Events (SAEs). Based on preclinical studies and Phase I clinical trial, Granexin™ Gel with 100 μM concentration of the ACT1 peptide was chosen for Phase II evaluation in the treatment of VLUs.

For each subject, Granexin™ Gel (100 μM ACT1 peptide) was applied at Days 0 and 3, and Weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

A total of 101 subjects with VLU were screened. Of these, 5 (4.9%) were screen failures and 4 (4.04%) subjects fulfilled the inclusion/exclusion criteria but did not come for the Baseline visit. The remaining 92 (91.1%) subjects were enrolled and randomized in the study. A total of 15 (16.3%) subjects met exclusion criteria number 1 or did not have adequate evaluable wound photographic data post randomization and were excluded from the mITT and PP population.

A total of 70 (76.1%) subjects completed the study and the remaining 22 (23.9%) subjects did not complete the study of which 14 (63.6%) withdrew their consent, 4 (18.2%) showed protocol non-compliance, 3 (13.6%) were lost to follow up, and 1 (4.5%) subject (Subject 1115; SoC group) reported death due to Myocardial infarction (Table 30.

TABLE 30 Disposition of Subjects Treatment Group Granexin ™ Gel plus SoC SoC Overall Category (N = 46) (N = 46) (N = 92) Population, n (%)^([1]) Screened 101 Randomized 46 (100.0%) 46 (100.0%) 92 (100.0%) Intent-to-treat 46 (100.0%) 46 (100.0%) 92 (100.0%) Modified Intent-to-treat 41 (89.1%)  36 (78.3%)  77 (83.7%)  Per-Protocol 35 (76.1%)  33 (71.7%)  68 (73.9%)  Safety 46 (100.0%) 46 (100.0%) 92 (100.0%) Total number of subjects 34 (73.9%)  36 (78.3%)  70 (76.1%)  completed the study Total number of subjects 12 (26.1%)  10 (21.7%)  22 (23.9%)  not completed the study Reason for discontinuation Consent Withdrawn 10 (83.3%)  4 (40.0%) 14 (63.6%)  Death 0 (0.0%)  1 (10.0%) 1 (4.5%)  Lost to follow up 2 (16.7%) 1 (10.0%) 3 (13.6%) Protocol non-compliance 0 (0.0%)  4 (40.0%) 4 (18.2%) Note: ^([1])Percentage was calculated taking respective column header group count as denominator.

The following data sets were analyzed:

ITT Population:

The ITT population included all subjects randomized to any treatment group, received at least one dose of study medication or reference treatment, and performed study assessments within and/or outside the time window specified in the protocol.

mITT Population:

The mITT population excluded all subjects that met exclusion criterion 1 or did not have adequate evaluable wound photographic data post randomization

PP Analysis Population:

The PP analysis population was redefined to include subjects who did not have any major protocol violation.

Safety Population:

The safety population included all subjects who were randomized to either of the treatment groups in the study.

The ITT population included 92 subjects; the mITT population included 77 subjects, PP population included 68 subjects; and the safety population included 92 subjects.

Modified Intent-to-Treat Population: The following subjects were excluded from the mITT population:

Granexin™ Gel Plus SoC Group:

Subject 304, Subject 501, Subject 503, Subject 505, and Subject 906

SoC Group:

Subject 103, Subject 104, Subject 1004, Subject 1006, Subject 1008, Subject 1017, Subject 1022, Subject 1026, Subject 1027, and Subject 1031

Per Protocol Population: The following subjects were excluded from the PP population:

Meeting exclusion criterion—Decrease or increase in the ulcer size by 30% or more during 7 day screening period or did not have adequate evaluable wound photographic data post randomization (15 subjects)

Granexin™ Gel Plus SoC Group:

Subject 304, Subject 501, Subject 503, Subject 505, and Subject 906

SoC Group:

Subject 103, Subject 104, Subject 1004, Subject 1006, Subject 1008, Subject 1017, Subject 1022, Subject 1026, Subject 1027, and Subject 1031

Wound Photograph Data not Available (9 Subjects)

Granexin™ Gel Plus SoC Group:

Subject 303, Subject 904, Subject 908, Subject 914, Subject 917, and subject 1102

SoC Group:

Subject 502, Subject 1109, and Subject 1116

Demographics and Baseline Characteristics

Of 92 enrolled subjects, 83 (90.2%) were males and 9 (9.8%) were females. The mean±SD age was 49.8±12.71 years, weight was 70.47±14.89 kg, height 168.35±7.19 cm, and BMI was 24.79±4.55 kg/m², respectively. Ten (10.9%) subjects had a smoking habit with the mean±SD number of cigarettes smoked per day as 8.8±7.33.

TABLE 31 Summary of Subject Demographics at Screening Treatment Group Granexin ™ Gel Statistics/ plus SoC SoC Overall Category, n (%)^([1]) (N = 46) (N = 46) (N = 92) Age (years)^([2]) n 46 46 92 Mean 48.2 51.5 49.8 SD 12.47 12.87 12.71 Median 48.0 53.0 51.0 Range (Min.:Max.) (18:71) (22:79) (18:79) Gender Male 44 (95.7) 39 (84.8) 83 (90.2%) Female 2 (4.3)  7 (15.2) 9 (9.8%) Weight (kg)^([3]) n 46 46 92 Mean 72.15 68.79 70.47 SD 15.014 14.739 14.891 Median 69.40 67.60 69.00 Range (Min.:Max.)  (48.0:118.0)  (40.6:110.0)  (40.6:118.0) Height (cm)^([4]) n 46 46 92 Mean 169.38 167.32 168.35 SD 5.918 8.223 7.199 Median 171.00 168.00 169.85 Range (Min.:Max.) (156.0:180.0) (148.0:186.2) (148.0:186.2) Body Mass Index (kg/m²)^([5]) n 46 46 92 Mean 25.114 24.478 24.796 SD 4.7553 4.3794 4.5572 Median 23.930 23.807 23.807 Range (Min.:Max.) (16.04:38.97) (16.06:34.94) (16.04:38.97) Smoking Habits Yes 4 (8.7)  6 (13.0) 10 (10.9%) No 42 (91.3)    40 (87.0%) 82 (89.1%) If yes, Current 3 (6.5)  5 (10.9) 8 (8.7%) Past 1 (2.2) 1 (2.2) 2 (2.2%) Duration of Smoking (months) n 4 6 10 Mean 300.0 345.7 327.4 SD 48.99 53.52 54.29 Median 300.0 330.0 300.0 Range (Min.:Max.) (240:360) (300:420) (240:420) Missing 0 0 0 No. of Cigarettes Smoked per day n 3 5 8 Mean 8.8 8.8 8.8 SD 9.67 6.87 7.33 Median 3.5 6.0 6.0 Range (Min.:Max.)  (3:20)  (2:20)  (2:20) Missing 1 1 2 Note: ^([1])Percentages are calculated taking respective column header group count as denominator. ^([2])Calculated age as: Age = [(Informed consent signed Date (Screening) − Date of Birth + 1)/365.25]. ^([3])Calculated Weight as: lbs/2.2 = kilograms. ^([4])Calculated Height as: (Height (cms) = Height in inches * 2.538). ^([5])Calculated BMI as: {Weight (kg)/(Height (m))² }.

Ulcer Identification History

Thirty (32.6%) subjects reported wound location on medial side of leg followed by 20 (21.7%) subjects each reporting on anterior side of leg and lateral side of leg. The overall mean±SD duration of wound history was 68.4±149.65 weeks and mean wound area was 353.64±367.196 mm², respectively (Table 32).

TABLE 32 Summary of Ulcer Identification History Treatment Group Granexin ™ Gel Statistics/ plus SoC SoC Overall Category, n (%)^([1]) (n = 46) (n = 46) (N = 92) Wound Location Anteromedial 0 (0.0) 1 (2.2)  1 (1.1) Anterior side of leg 11 (23.9) 9 (19.6) 20 (21.7) Lateral Malleolus 2 (4.3) 6 (13.0) 8 (8.7) Lateral Side of leg 12 (26.1) 8 (17.4) 20 (21.7) Medial Malleolus 3 (6.5) 5 (10.9) 8 (8.7) Medial side of leg 17 (37.0) 13 (28.3)  30 (32.6) Posterior side of leg 1 (2.2) 4 (8.7)  5 (5.4) Duration of Wound History (weeks) n 46 46 92 Mean 62.7 74.0 68.4 SD 119.46 175.92 149.65 Median 16.0 16.0 16.0 Range (Min.:Max.) (4:511) (4:788) (4:788) Wound Area (mm²) (Length * Width) n 41 43 84 Mean 347.47 359.51 353.64 SD 369.266 369.484 367.196 Median 236.43 233.43 234.93 Range (Min.:Max.) (19.0:1804.9) ( 0.0:1576.8) ( 0.0:1804.9) Note: ^([1])Percentages are calculated taking respective column header group count as denominator.

Primary Efficacy Endpoint: Mean Percent Wound Closure from Baseline to Week 12

Table 33 summarizes the parametric analysis of the mean percent reduction of wound area (in mm²) from Baseline to Week 12 in the ITT population. Of the 92 subjects in the ITT population, non-missing observations from Baseline to Week 12 for percent reduction of wound area were available for 62 subjects. The mean percent±SD reduction of wound area from Baseline to Week 12 was not statistically significantly higher in Granexin™ Gel plus SoC group than SoC group (Granexin™ Gel plus SoC group: 79.28%±50.37; SoC group: 36.28%±179.78; p-value=0.0741) as per parametric analysis. Similar results were obtained after sensitivity analysis (p-value=0.0921). The model factors such as treatment, visit, treatment*visit, and ankle circumference were significantly related to the mean percent reduction in wound area from Baseline to Week 12 for the ITT population (Treatment: p-value=0.0062; Visit: p-value<0.0001; Treatment*Visit p-value=0.0139; Ankle Circumference: p-value=0.0200).

FIG. 10 displays the longitudinal response profile of the mean percent reduction of wound area at all visits by treatment group of the ITT population.

TABLE 33 Mean Percent Reduction of Wound Area (mm²) from Baseline to Week 12 (Parametric analysis) ITT Population (N = 92) Treatment Group Granexin ™ Gel plus SoC SoC Statistics (N = 46) (N = 46) Total number of N = 62^([1]) subjects evaluated Percent reduction of wound area from Baseline to Week 12 (mm²) n 34 28 Mean 79.279 36.282 SD 50.3695 179.7788 Median 100.000 93.320 Range (Min.:Max.) (−68.85:100.00) (−849.50:100.00) LS Mean Estimate 73.2182 53.0164 Difference Estimate^([2]) 20.2018 SE^([3]) 11.2336 95% CI (L:U.) (−1.99:42.39) p-value^([4],[5]) 0.0741 Model factor^([4] [5]) p-value Treatment 0.0062 Visit <0.0001 Treatment*Visit 0.0139 Exudate level 0.1440 Viable tissue 0.6703 Strata 0.1532 Ankle circumference 0.0200 Wound duration 0.5371 BMI 0.7303 Note: ^([1])“Total no. of subjects evaluated” represented subjects having non-missing observations for change from Baseline to Week 12 for percent reduction of wound area ^([2])Difference estimate for Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plus SoC)-SoC). ^([3])SE of Granexin ™ Gel plus SoC indicated Standard Error of Differences (Granexin ™ Gel plus SoC)-SoC). ^([4])p-value for Granexin ™ Gel plus SoC indicated significance of treatment differences (Granexin ™ Gel plus SoC)-(SoC). ^([5])ANCOVA mixed model with repeated measures was used to compare average percent reduction in wound area with treatment, visit and treatment*visit as factors and strata, wound duration, viable tissue, exudate level and the BMI as covariates using PROC Mixed procedure of SAS software.

Table 34 summarizes the parametric analysis of the mean percent reduction of wound area (in mm²) from Baseline to Week 12 in the mITT population. Of the 77 subjects in the mITT population, non-missing observations from Baseline to Week 12 for percent reduction of wound area were available for 59 subjects. The mean percent±SD reduction of wound area from Baseline to Week 12 was not statistically significantly higher in Granexin™ Gel plus SoC group than SoC group (Granexin™ Gel plus SoC group: 78.96%±51.11; SoC group: 31.38%±185.89; p-value=0.0643) as per parametric analysis. Similar results were obtained after sensitivity analysis (p-value=0.0785). The model factors such as treatment, visit, treatment*visit, and ankle circumference were significantly related to the mean percent reduction in wound area from Baseline to Week 12 for the mITT population (Treatment: p-value=0.0028; Visit: p-value<0.0001; Treatment*Visit p-value=0.0424; Ankle Circumference: p-value=0.0428).

Error! Reference source not found. 11 displays the longitudinal response profile of the mean percent reduction of wound area at all visits by treatment group of the mITT population.

TABLE 34 Mean Percent Reduction of Wound Area (mm²) from Baseline to Week 12 (Parametric analysis) mITT Population (N = 77) Treatment Group Granexin ™ Gel plus SoC SoC Statistics (N = 41) (N = 36) Total number of N = 59^([1]) subjects evaluated Percent reduction of wound area from Baseline to Week 12 (mm²) n 33 26 Mean 78.957 31.380 SD 51.1148 185.8931 Median 100.000 87.352 Range (Min.:Max.) (−68.85:100.00) (−849.50:100.00) LS Mean Estimate 73.275 51.921 Difference Estimate^([2]) 21.3543 SE^([3]) 11.4565 95% CI (L:U.) (−1.28:43.99) p-value^([4],[5]) 0.0643 Model factor^([4] [5]) p-value Treatment 0.0028 Visit <.0001 Treatment * Visit 0.0424 Exudate level 0.1232 Viable tissue 0.7318 Strata 0.1592 Ankle circumference 0.0428 Wound duration 0.5846 BMI 0.6308 Note: ^([1])“Total no. of subjects evaluated” represented subjects having non-missing observations for change from Baseline to Week 12 for percent reduction of wound area ^([2])Difference estimate for Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plus SoC)-SoC). ^([3])SE of Granexin ™ Gel plus SoC indicated Standard Error of Differences (Granexin ™ Gel plus SoC)-SoC). ^([4])p-value for Granexin ™ Gel plus SoC indicated significance of treatment differences (Granexin ™ Gel plus SoC)-(SoC). ^([5])ANCOVA mixed model with repeated measures was used to compare average percent reduction in wound area with treatment, visit and treatment * visit as factors and strata, wound duration, viable tissue, exudate level and the BMI as covariates using PROC Mixed procedure of SAS software.

The parametric analysis of the mean percent reduction of wound area (in mm²) from Baseline to Week 12 in the PP population is summarized in Table 35 Of the 68 subjects in the PP population, non-missing observations for percent reduction of wound area from Baseline to Week 12 were available for 58 subjects. The mean percent±SD reduction of wound area from Baseline to Week 12 was not statistically significantly higher in Granexin™ Gel plus SoC group than in SoC group (Granexin™ Gel plus SoC group: 78.96%±51.11; SoC group: 28.63%±189.18) (p-value=0.0722) as per parametric analysis. Similarly, on applying LOCF approach for imputation of missing values, results obtained after sensitivity analysis, were not statistically significant (p-value=0.1151. The model factors such as treatment, visit, treatment*visit, and ankle circumference were significantly related to the mean percent reduction in wound area from Baseline to Week 12 for the PP population (Treatment: p-value=0.0025; Visit: p-value<0.0001; Treatment*visit p-value=0.0228; and Ankle Circumference p-value=0.0350). Error! Reference source not found. 12 displays the longitudinal response profile of the mean percentage reduction of wound area at all visits by treatment group of the PP population.

TABLE 35 Mean Percent Reduction of Wound Area (mm²) from Baseline to Week 12 (Parametric analysis) PP Population (N = 68) Treatment Group Granexin ™ Gel plus SoC SoC Statistics (N = 35) (N = 33) Total no. of subjects evaluated N = 58^([1]) Percent reduction of wound area from Baseline to Week 12 n 33 25 Mean 78.957 28.635 SD 51.1148 189.1879 Median 100.000 85.225 Range (Min.:Max.) (−68.85:100.00) (−849.50:100.00) LS mean estimate 74.5294 53.5060 Difference estimate^([2]) 21.0234 SE^([3]) 11.6048 95% CI (L.:U.) (−1.92:43.97) p-value^([4],[5]) 0.0722 Model factor^([3] [4]) p-value Treatment 0.0025 Visit <0.0001 Treatment * Visit 0.0228 Exudate level 0.1306 Viable tissue 0.7206 Strata 0.2849 Ankle circumference 0.0350 Wound duration 0.5391 Body mass index 0.5793 Note: ^([1])“Total no. of subjects evaluated” represented subjects having non-missing observations for dependent variable ^([2])Difference estimate for Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plus SoC)-SoC). ^([3])SE of Granexin ™ Gel plus Standard of Care indicated Standard Error of Differences (Granexin ™ Gel plus SoC)-SoC). ^([4])p-value for Granexin ™ Gel plus SoC indicated significance of treatment differences (Granexin ™ Gel plus SoC)-(SoC). ^([5])ANCOVA mixed model with repeated measures was used to compare average percentage reduction in wound area with treatment, visit and treatment * visit as factors and strata, wound duration, viable tissue, exudate level and the body mass index as covariates using PROC Mixed procedure of SAS software.

To confirm the applicability of parametric statistical inference methodology in the wound reduction analysis, the data was tested to determine if it followed normal distribution. Using PROC UNIVARIATE of SAS® (version 9.1.3) Shapiro-Wilk W statistic was calculated. The p-value (<0.0001) indicated that the data did not follow a normal distribution. This was further confirmed by the Q-Q plot for both the ITT and PP populations in which the observations were not in a straight line and did not show a normal distribution. Therefore, the hypothesis of normal distribution of the data in the sample was rejected and non-parametric analysis was conducted.

Table 36 summarizes the non-parametric analysis for mean percent reduction of wound area from Baseline to Week 12 in the ITT population. The Wilcoxon Mann-Whitney U test is a non-parametric test which determines whether there is a difference in two samples of independent observations. In Granexin™ Gel plus SoC group, the actual sum of scores (1210.00) was found higher than the expected sum of scores under the null hypothesis (1071.00). In SoC group, the actual sum of scores (743.00) was found lower than the expected sum of the scores under the null hypothesis (882.00). The actual sum of scores was calculated by adding the ranks of both groups after sorting data of wound size reduction in ascending order and the expected sum of scores was calculated as the sum of scores when there is no difference between the 2 groups. Subjects in Granexin™ Gel plus SoC group had statistically significantly higher percent reduction of wound area from Baseline to Week 12 than in SoC group (p-value=0.0238).

TABLE 36 Mean Percent Reduction of Wound Area (mm²) from Baseline to Week 12 (Non-parametric analysis) ITT Population (N = 92) Treatment Group Granexin ™ Gel plus SoC SoC Statistics (N = 46) (N = 46) Percent reduction of wound area from Baseline to Week 12 Sum of score 1210.00 743.00 Expected under H_(o) 1071.00 882.00 p-value^([1]) 0.0238 Note: ^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group. General Note: Mean percentage reduction from Baseline = ((Baseline − week X)/Baseline) * 100 H_(o): Null Hypothesis If wound was healed and all further visits were missing then 100% reduction had been carried forward to missing visits.

Table 37 summarizes the non-parametric analysis for mean percent reduction of wound area from Baseline to Week 12 in the mITT population. The Wilcoxon Mann-Whitney U test is a non-parametric test which determines whether there is a difference in two samples of independent observations. In Granexin™ Gel plus SoC group, the actual sum of scores (1136.00) was found higher than the expected sum of scores under the null hypothesis (990.00). In SoC group, the actual sum of scores (634.00) was found lower than the expected sum of the scores under the null hypothesis (780.00). The actual sum of scores was calculated by adding the ranks of both groups after sorting data of wound size reduction in ascending order and the expected sum of scores was calculated as the sum of scores when there is no difference between the 2 groups. Subjects in Granexin™ Gel plus SoC group had statistically significantly higher percent reduction of wound area from Baseline to Week 12 than in SoC group (p-value=0.0105).

TABLE 37 Mean Percent Reduction of Wound Area (mm²) from Baseline to Week 12 (Non-parametric analysis) mITT Population (N = 77) Treatment Group Granexin ™ Gel plus SoC SoC Statistics (N = 41) (N = 36) Percent reduction of wound area from Baseline to Week 12 Sum of score 1136.00 634.00 Expected under H_(o) 990.00 780.00 p-value^([1]) 0.0105 Note: ^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group. General Note: Mean percentage reduction from Baseline = ((Baseline − week X)/Baseline) * 100 H_(o): Null Hypothesis If wound was healed and all further visits were missing then 100% reduction had been carried forward to missing visits.

Table 38 summarizes the non-parametric analysis for mean percent reduction of wound area from Baseline to Week 12 in the PP population. In Granexin™ Gel plus SoC group, the actual sum of scores (1123.00) was found higher than the expected sum of scores under the null hypothesis (973.50) and in SoC group, the actual sum of scores (588.00) was found lower than the expected sum of the scores under the null hypothesis (737.50). Subjects in Granexin™ Gel plus SoC group had statistically significantly higher percent reduction of wound area from Baseline to Week 12 than subjects in SoC group (p-value=0.0073).

TABLE 38 Mean Percent Reduction of Wound Area (mm²) from Baseline to Week 12 (Non-parametric analysis) PP Population (N = 68) Treatment Group Granexin ™ Gel plus SoC group SoC group Statistics (N = 35) (N = 33) Percent reduction of wound area from Baseline to Week 12 Sum of score 1123.00 588.00 Expected under H_(o) 973.50 737.50 p-value^([1]) 0.0073 Note: ^([1])p-value was evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group.

Secondary Analysis: Mean Percentage Reduction of Wound Area from Baseline to Week 4

The parametric evaluation of mean percent wound closure from Baseline to Week 4 in the ITT population is summarized in Table 39. Of 92 subjects in the ITT population, non-missing observations for percent reduction of wound area from Baseline to Week 4 were available for a total of 56 subjects. The mean percent±SD reduction of wound area from Baseline to Week 4 was statistically significantly higher in Granexin™ Gel plus SoC group (76.99±29.74) than in SoC group (39.080±42.80) (p-value=0.0040). The model factors such as treatment, visit, and ankle circumference were significantly related to the mean percent reduction of wound area from Baseline to Week 4 in the ITT population (treatment p-value=0.0062; Visit p-value<0.0001; treatment*visit p-value=0.0139; and Ankle Circumference p-value=0.0200).

TABLE 39 Mean Percent Reduction of Wound Area (mm²) from Baseline to Week 4 (Parametric analysis) ITT Population (N = 92) Treatment Group Granexin ™ Gel plus SoC SoC Statisctics (N = 46) (N = 46) Total no. of subjects evaluated N = 56^([1]) Percent reduction of wound area from Baseline to Week 4 n 30 26 Mean 76.996 39.080 SD 29.7470 42.8061 Median 97.369 41.872 Range (Min.:Max.)  (0.40:100.00) (−35.54:100.00) LS mean estimate 60.2593 27.0027 Difference estimate [2] 33.2566 SEB [3] 11.3883 95% CI (L.:U.) (10.76:55.75) p-value [4], [5] 0.0040 Model Factor^([3] [4]) p-value Treatment 0.0062 Visit <0.0001 Treatment * Visit 0.0139 Exudate level 0.1440 Viable tissue 0.6703 Strata 0.1532 Ankle Circumference 0.0200 Wound duration 0.5371 Body mass index 0.7303 Note: ^([1])“Total no. of subjects evaluated” represented subjects having non-missing observations for dependent variable [2] Difference estimate for Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plus SoC)-SoC). [3] SE of Granexin ™ Gel plus SoC indicated Standard Error of Differences (Granexin ™ Gel plus SoC)-SoC). [4] p-value for Granexin ™ Gel plus SoC indicated significance of treatment differences (Granexin ™ Gel plus SoC)-(SoC). [5] ANCOVA mixed model with repeated measures was used to compare average percentage reduction in wound area with treatment, visit and treatment * visit as factors and strata, wound duration, viable tissue, exudate level and the body mass index as covariates using PROC Mixed procedure of SAS software.

The parametric evaluation of mean percent wound closure from Baseline to Week 4 in the mITT population is summarized in Table 40. Of 77 subjects in the mITT population, non-missing observations for percent reduction of wound area from Baseline to Week 4 were available for a total of 54 subjects. The mean percent±SD reduction of wound area from Baseline to Week 4 was statistically significantly higher in Granexin™ Gel plus SoC group (76.99±29.74) than in SoC group (34.00±40.52) (p-value=0.0022). The model factors such as treatment, visit, and ankle circumference were significantly related to the mean percent reduction of wound area from Baseline to Week 4 in the mITT population (treatment p-value=0.0028; Visit p-value<0.0001; treatment*visit p-value=0.0424; and Ankle Circumference p-value=0.0428).

TABLE 40 Mean Percent Reduction of Wound Area (mm²) from Baseline to Week 4 (Parametric analysis) mITT Population (N = 77) Treatment Group Granexin ™ Gel plus SoC SoC Statistics (N = 41) (N = 36) Total no. of subjects evaluated N = 54^([1]) Percent reduction of wound area from Baseline to Week 4 n 30 24 Mean 76.996 34.003 SD 29.7470 40.5227 Median 97.369 38.217 Range (Min.:Max.)  (0.40:100.00) (−35.54:100.00) LS mean estimate 60.394 24.421 Difference estimate [2] 35.9729 SE [3] 11.5191 95% CI (L.:U.) (13.21:58.73) p-value [4], [5] 0.0022 Model Factor^([3] [4]) p-value Treatment 0.0028 Visit <0.0001 Treatment * Visit 0.0424 Exudate level 0.1232 Viable tissue 0.7318 Strata 0.1592 Ankle Circumference 0.0428 Wound duration 0.5846 Body mass index 0.6308 Note: ^([1])“Total no. of subjects evaluated” represented subjects having non-missing observations for dependent variable [2] Difference estimate for Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plus SoC)-SoC). [3] SE of Granexin ™ Gel plus SoC indicated Standard Error of Differences (Granexin ™ Gel plus SoC)-SoC). [4] p-value for Granexin ™ Gel plus SoC indicated significance of treatment differences (Granexin ™ Gel plus SoC)-(SoC). [5] ANCOVA mixed model with repeated measures was used to compare average percentage reduction in wound area with treatment, visit and treatment * visit as factors and strata, wound duration, viable tissue, exudate level and the body mass index as covariates using PROC Mixed procedure of SAS software.

The parametric analysis of mean percentage reduction of wound area from Baseline to Week 4 in the PP population is summarized in Table 41. Of 68 subjects in the PP population, non-missing observations for change from Baseline to Week 4 for percent reduction of wound area were available for a total of 53 subjects. The mean percent±SD reduction of wound area (mm²) from Baseline to Week 4 was statistically significantly higher in Granexin™ Gel plus SoC group subjects (76.99±29.74) than in SoC group subjects (31.13±38.86) (p-value=0.0012). The model factors such as treatment, visit, treatment*visit, and ankle circumference were significantly related to the mean percent reduction of wound area from Baseline to Week 4 in the PP population (treatment p-value=0.0025; Visit p-value<0.0001; treatment*visit p-value=0.0228; and Ankle Circumference p-value=0.0350).

TABLE 41 Mean Percent Reduction of Wound Area (mm²) from Baseline to Week 4 (Parametric analysis) PP Population (N = 68) Treatment Group Granexin ™ Gel plus SoC SoC Statistics (N = 35) (N = 33) Total no. of subjects evaluated N = 53^([1]) Percent reduction of wound area from Baseline to Week 4 n 30 23 Mean 76.996 31.134 SD 29.7470 38.8605 Median 97.369 34.902 Range (Min.:Max.)  (0.40:100.00) (−35.54:100.00) LS Mean Estimate 62.3396 23.8304 Difference Estimate^([2]) 38.5092 SE^([3]) 11.6027 95% CI (L.:U.) (15.56:61.46) p-value^([4],[5]) 0.0012 Model Factor^([3] [4]) p-value Treatment 0.0025 Visit <0.0001 Treatment * Visit 0.0228 Exudate level 0.1306 Viable tissue 0.7206 Strata 0.2849 Ankle circumference 0.0350 Wound duration 0.5391 Body mass index 0.5793 Note: ^([1])“Total no. of subjects evaluated” represented subjects having non-missing observations for dependent variable ^([2])Difference estimate for Granexin ™ Gel plus SoC indicated (Granexin ™ Gel plus SoC)-SoC). ^([3])SE of Granexin ™ Gel plus SoC indicated Standard Error of Differences (Granexin ™ Gel plus SoC)-SoC). ^([4])p-value for Granexin ™ Gel plus SoC indicated significance of treatment differences (Granexin ™ Gel plus SoC)-(SoC). ^([5])ANCOVA mixed model with repeated measures was used to compare average percentage reduction in wound area with treatment, visit and treatment * visit as factors and strata, wound duration, viable tissue, exudate level and the body mass index as covariates using PROC Mixed procedure of SAS software.

42 summarizes the non-parametric analysis for mean percent reduction of wound area from Baseline to Week 4 in the ITT population. In Granexin™ Gel plus SoC group, the actual sum of scores (1060.00) was found higher than the expected sum of scores under the null hypothesis (855.00). In SoC group, the actual sum of scores (536.00) was found lower than the expected sum of the scores under the null hypothesis (741.00). Granexin™ Gel plus SoC group subjects had statistically significantly higher percent reduction of wound area than SoC group subjects from Baseline to Week 4 (p-value=0.0006).

TABLE 42 Mean Percent Reduction of Wound Area (mm²) from Baseline to Week 4 (Non-parametric analysis) ITT Population (N = 92) Treatment Group Granexin ™ Gel plus SoC SoC Statistics (N = 46) (N = 46) Percent reduction of wound area from Baseline to Week 4 Sum of score 1060.00 536.00 Expected under H_(o) 855.00 741.00 p-value^([1]) 0.0006 Note: ^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group.

Table 43 summarizes the non-parametric analysis for mean percent reduction of wound area from Baseline to Week 4 in the mITT population. In Granexin™ Gel plus SoC group, the actual sum of scores (1046.00) was found higher than the expected sum of scores under the null hypothesis (825.00). In SoC group, the actual sum of scores (439.00) was found lower than the expected sum of the scores under the null hypothesis (660.00). Granexin™ Gel plus SoC group subjects had statistically significantly higher percent reduction of wound area than SoC group subjects from Baseline to Week 4 (p-value=0.0001).

TABLE 43 Mean Percent Reduction of Wound Area (mm²) from Baseline to Week 4 (Non-parametric analysis) mITT Population (N = 77) Treatment Group Granexin ™ Gel plus SoC SoC Statistics (N = 41) (N = 36) Percent reduction of wound area from Baseline to Week 4 Sum of score 1046.00 439.00 Expected under H_(o) 825.00 660.00 p-value^([1]) 0.0001 Note: ^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group.

The non parametric analysis for mean percent reduction of wound area from Baseline to Week 4 in the PP population is summarized in Table 44. In Granexin™ Gel plus SoC group, the actual sum of scores (1039.00) was found higher than the expected sum of scores under the null hypothesis (810.00). In SoC group, the actual sum of scores (392.00) was found lower than the expected sum of the scores under the null hypothesis (621.00). Granexin™ Gel plus SoC group subjects had statistically significantly higher percent reduction of wound area than SoC group subjects from Baseline to Week 4 (p-value<0.0001).

TABLE 44 Mean Percent Reduction of Wound Area (mm²) from Baseline to Week 4 (Non-parametric analysis) PP Population (N = 68) Treatment Group Granexin ™ Gel plus SoC SoC Statistics (N = 35) (N = 33) Percent reduction of wound area from Baseline to Week 4 Sum of score 1039.00 392.00 Expected under H_(o) 810.00 621.00 p-value^([1]) <0.0001 Note: ^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group.

Time to 100% Wound Closure: Time taken for first complete (100%) wound closure for the ITT population is summarized in Table 45 and FIG. 13 Complete wound closure was defined as 100% epithelialization of the wound and absence of any drainage from the wound. Out of 92 subjects, 28 [60.9%] subjects in the Granexin™ Gel plus SoC group and 15 (32.6%) subjects in the SoC group had 100% wound closure by Week 12. The median duration of 100% wound closure was 6 weeks (90% CI: 4 to 8 weeks) in Granexin™ Gel plus SoC group and 12.14 weeks (90% CI: 10.14 to not achieved) in SoC group. Subjects in Granexin™ Gel plus SoC group took statistically significantly less time to achieve 100% wound closure than SoC group subjects (p-value=0.0006). Five subjects (Granexin™ Gel plus SoC group: 4; SoC group: 1) having missing value after Screening or having missing visit date were not included in the analysis. The remaining 44 subjects (Granexin™ Gel plus SoC group: 14 [30.4%]; SoC group: 30 [65.2%]) were censored due to reasons such as subjects not having complete wound closure, lack of photographic evaluation data, withdrawal of consent, loss to follow-up, discontinued due to protocol non compliance, or death.

TABLE 45 Summary of Time to First Complete (100%) Wound Closure by Week 12 ITT Population (N = 92) Treatment Group Granexin ™ Gel plus SoC SoC Statistics^([1]) (N = 46) (N = 46) Number (%) of subjects with 28 (60.9%) 15 (32.6%) complete (100%) wound closure Number (%) of censored 14 (30.4%) 30 (65.2%) subjects Median duration (weeks) of 6.00 (4.00; 8.00) 12.14 (10.14; NA) complete wound closure (90% CI)^([2]) p-value^([3]) 0.0006 Note: ^([1])Percentage was calculated taking count of corresponding treatment groups as denominator. ^([2])The median duration of complete wound closure was estimated by Kaplan-Meier method and also 90% CI was calculated for the median duration of 100% wound closure. ^([3])p-value was calculated using log-rank test for treatment groups using Proc Lifetest procedure of SAS software.

Time taken for first complete (100%) wound closure for the mITT population is summarized in Table 46 and FIG. 14 Error! Reference source not found. Complete wound closure was defined as 100% epithelialization of the wound and absence of any drainage from the wound. Out of 77 subjects, 28 (68.3%) subjects in the Granexin™ Gel plus SoC group and 15 (36.1%) subjects in the SoC group had 100% wound closure by Week 12. The median duration of 100% wound closure was 6 weeks (90% CI: 4 to 7 weeks) in Granexin™ Gel plus SoC group and not achieved (90% CI: 12 to not achieved) in SoC group. Subjects in Granexin™ Gel plus SoC group took statistically significantly less time to achieve 100% wound closure than SoC group subjects (p-value<0.0001). Five subjects (Granexin™ Gel plus SoC group: 4; SoC group: 1) having missing value after Screening or having missing visit date were not included in the analysis. The remaining 34 subjects (Granexin™ Gel plus SoC group: 11 (26.8%); SoC group: 23 (63.9%) were censored due to reasons such as subjects not having complete wound closure, lack of photographic evaluation data, withdrawal of consent, loss to follow-up, discontinued due to protocol non compliance, or death.

TABLE 46 Summary of Time to First Complete (100%) Wound Closure by Week 12 mITT Population (N = 77) Treatment Group Granexin ™ Gel plus SoC SoC Statistics^([1]) (N = 41) (N = 36) Number (%) of subjects 28 (68.3%) 13 (36.1%) with complete (100%) wound closure Number (%) of censored 11 (26.8%) 23 (63.9%) subjects Median duration (weeks) of 6.00 (4.00; 7.00) NA (12.00; NA)^([3]) complete wound closure (90% CI)^([2]) p-value^([4]) <0.0001 Note: ^([1])Percentage was calculated taking count of corresponding treatment groups as denominator. ^([2])The median duration of complete wound closure was estimated by Kaplan-Meier method and also 90% CI was calculated for the median duration of 100% wound closure. ^([3])NA: Not achieved ^([4])p-value was calculated using log-rank test for treatment groups using Proc Lifetest procedure of SAS software.

Time taken for 100% wound closure in the PP population is summarized in Table 47 and FIG. 15. Out of 68 subjects, 40 (Granexin™ Gel plus SoC group: 28 [80.0%]; SoC group: 12 [36.4%]) had 100% wound closure by Week 12. The median duration of 100% wound closure was 6 weeks (90% CI was 4 to 7 weeks) in Granexin™ Gel plus SoC group and median duration was not achieved (90% CI: 12.00 to NA) in 12 weeks of treatment evaluation in SoC group. Subjects in Granexin™ Gel plus SoC group took statistically significantly less time to achieve 100% wound closure than SoC group subjects (p-value<0.0001). A total of 28 subjects (Granexin™ Gel plus SoC group: 7[20.0%]; SoC: 21 (63.6%]) were censored due to reasons such as subjects not having complete wound 100% closure, lack of photographic evaluation data, withdrawal of consent, loss to follow-up, discontinued due to protocol non compliance, or death.

TABLE 47 Summary of Time to First Complete (100%) Wound Closure by Week 12 PP Population (N = 68) Granexin ™ Gel plus SoC SoC Statistics^([1]) (N = 35) (N = 33) Number (%) of subjects with 28 (80.0%) 12 (36.4%) complete (100%) wound closure Number (%) of censored 7 (20.0%) 21 (63.6%) subjects Median duration (weeks) of 6.00 (4.00; 7.00) NA (12.00; NA) complete wound closure (90% CI)^([2]) p-value^([3]) <0.0001 Note: ^([1])Percentage was calculated taking count of corresponding treatment groups as denominator. ^([2])The median duration of complete wound closure was estimated by Kaplan-Meier method and also 90% CI was calculated for the median duration of 100% wound closure. ^([3])p-value was calculated using Log-rank test for treatment groups using Proc Lifetest procedure of SAS software. Time taken to achieve 50% wound closure in the ITT population is summarized in Table 48 and FIG. 16. Out of 92 subjects, 61 (Granexin™ Gel plus SoC group: 34 [73.9%]; SoC group: 25 [54.3%]) had 50% wound closure by Week 12. The median duration of 50% wound closure was 2.86 weeks (90% CI was 2.14 to 3 weeks) in Granexin™ Gel plus SoC group and 6.86 weeks (90% CI: 5.00 to 9.14) in SoC group. Subjects in Granexin™ Gel plus SoC group took statistically significantly less time to achieve 50% wound closure than SoC group subjects (p-value=0.0002). Five (5.5%) subjects having missing values after Screening or having missing visit dates were not included in the analysis. A total of 28 subjects (Granexin™ Gel plus SoC group: 8 [17.4%]; SoC group: 20 [43.5%]) were censored due to reasons such as subjects not having complete 50% wound closure, lack of photographic evaluation data, withdrawal of consent, loss to follow-up, discontinued due to protocol non compliance, or death.

TABLE 48 Summary of Time to First Complete 50% Wound Closure by Week 12 ITT Population (N = 92) Treatment Group Granexin ™ Gel plus SoC SoC Statistics^([1]) (n = 46) (n = 46) Number (%) of subjects with 34 (73.9%) 25 (54.3%) complete (50%) wound closure Number (%) of censored 8 (17.4%) 20 (43.5%) subjects Median duration (weeks) of 2.86 (2.14; 3.00) 6.86 (5.00; 9.14) complete wound closure (90% CI)^([2]) p-value^([3]) 0.0002 Note: ^([1])Percentage was calculated taking count of corresponding treatment groups as denominator. ^([2])The median duration of complete wound closure was estimated by Kaplan-Meier method and also 90% confidence interval was calculated for the median duration of 50% wound closure. ^([3])p-value was calculated using Log-rank test for treatment groups using Proc Lifetest procedure of SAS software

Time taken to achieve 50% wound closure in the mITT population is summarized in Table 49 and FIG. 17. Out of 77 subjects, 59 (Granexin™ Gel plus SoC group: 33 [80.5%]; SoC group: 23 [63.9%]) had 50% wound closure by Week 12. The median duration of 50% wound closure was 2.86 weeks (90% CI was 2.14 to 3 weeks) in Granexin™ Gel plus SoC group and 8 weeks (90% CI: 5.14 to 9.86) in SoC group. Subjects in Granexin™ Gel plus SoC group took statistically significantly less time to achieve 50% wound closure than SoC group subjects (p-value<0.0001). Five (5.5%) subjects having missing values after Screening or having missing visit dates were not included in the analysis. A total of 19 subjects (Granexin™ Gel plus SoC group: 6 [14.6%]; SoC group: 13 [36.1%]) were censored due to reasons such as subjects not having complete 50% wound closure, lack of photographic evaluation data, withdrawal of consent, loss to follow-up, discontinued due to protocol non compliance, or death.

TABLE 49 Summary of Time to First Complete 50% Wound Closure by Week 12 mITT Population (N = 77) Treatment Group Granexin ™ Gel plus SoC SoC Statistics^([1]) (n = 41) (n = 36) Number (%) of subjects with   33 (80.5%)   23 (63.9%) complete (50%) wound closure Number (%) of censored subjects   6 (14.6%)   13 (36.1%) Median duration (weeks) of 2.86 (2.00; 3.00) 8.00 (5.14; 9.86) complete wound closure (90% CI)^([2]) p-value^([3]) <0.0001 Note: ^([1])Percentage was calculated taking count of corresponding treatment groups as denominator. ^([2])The median duration of complete wound closure was estimated by Kaplan-Meier method and also 90% confidence interval was calculated for the median duration of 50% wound closure. ^([3])p-value was calculated using Log-rank test for treatment groups using Proc Lifetest procedure of SAS software.

The time taken to achieve 50% wound closure in the PP population is summarized in Table 50 and Error! Reference source not found. 18 Out of 68 subjects, 54 (Granexin™ Gel plus SoC group: 32 [91.4%]; SoC group: 22 [66.7%]) had 50% wound closure by Week 12. The median duration of 50% wound closure was 2.86 weeks (90% CI was 2 to 3 weeks) in Granexin™ Gel plus SoC group and 8 weeks (90% CI: 5.14 to 9.86) in SoC group. Subjects in Granexin™ Gel plus SoC group took statistically significantly less time to achieve 50% wound closure than SoC group subjects (p-value<0.0001). A total of 14 subjects (Granexin™ Gel plus SoC group: 3 [8.6%]; SoC: 11 [33.3%]) were censored as they did not complete 50% wound closure during the study period, or due to lack of photographic evaluation data, withdrawal of consent, loss to follow-up, discontinued due to protocol non compliance, or death.

TABLE 50 Summary of Time to First Complete (50%) Wound Closure by Week 12 PP Population (N = 68) Treatment Group Granexin ™ Gel plus SoC SoC Statistics^([1]) (N = 35) (N = 33) Number (%) of subjects with   32 (91.4%)   22 (66.7%) complete (50%) wound closure Number (%) of censored subjects   3 (8.6%)   11 (33.3%) Median duration (weeks) of 2.86 (2.00; 3.00) 8.00 (5.14; 9.86) complete wound closure (90% CI)^([2]) p-value^([3]) <0.0001 Note: ^([1])Percentage was calculated taking count of corresponding treatment groups as denominator. ^([2])The median duration of complete wound closure was estimated by Kaplan-Meier method and also 90% CI was calculated for the median duration of 50% wound closure. ^([3])p-value was calculated using Log-rank test for treatment groups using Proc Lifetest procedure of SAS software.

Additional to the Kaplan-Meier Model, Cox Proportional Hazards Regression Model was also used to compare the event-time distribution function between the two treatment groups. This analysis was conducted to evaluate if one or more covariates were associated with the time to 100% or 50% wound closure. Table 51 below summarizes the Cox Proportional Hazards Regression Analysis of time to complete wound closure in the ITT population. The effect of covariates estimated by the proportional hazards model is reported as hazard ratios. As per the hazards ratio and p-value, covariates such as treatment group and wound size showed an association with time to completion of wound closure (treatment group p-value=0.0406; wound size p-value=0.0265) and the subjects treated with Granexin™ Gel plus SoC would have wound closure 2.281 times more than the subjects treated with SoC only.

TABLE 51 Analysis of Time to First Complete (100%) Wound Closure (Cox's Proportional Hazards Regression Analysis) ITT Population (N = 92) 95% CI for Hazard Ratio Statistics Hazard Ratio^([1]) Lower Upper p-value Treatment group 2.281 1.036 5.021 0.0406 Wound size 0.998 0.997 1.000 0.0265 Wound duration 1.000 0.996 1.003 0.8431 Baseline wound depth 1.767 0.312 10.006 0.5197 BMI 1.003 0.922 1.092 0.9423 Note: ^([1])Cox Proportional Hazard ratio with 95% CI and p-value was calculated using PROC PHREG procedure.

Table 52 below summarizes the Cox Proportional Hazards Regression Analysis of time to complete wound closure in the mITT population. The effect of covariates estimated by the proportional hazards model is reported as hazard ratios. As per the hazards ratio and p-value, covariate such as treatment group showed an association with time to completion of wound closure (p-value=0.0139) and the subjects treated with Granexin™ Gel plus SoC would have wound closure 2.776 times more than the subjects treated with SoC only. Wound size, wound duration, baseline wound depth, and BMI were not associated with time to wound closure.

TABLE 52 Analysis of Time to First Complete (100%) Wound Closure (Cox's Proportional Hazards Regression Analysis) mITT Population (N = 77) 95% CI for Hazard Ratio Statistics Hazard Ratio^([1]) Lower Upper p-value Treatment group 2.776 1.230 6.265 0.0139 Wound size 0.998 0.997 1.000 0.0629 Wound duration 1.000 0.996 1.003 0.8838 Baseline wound depth 2.306 0.409 13.004 0.3439 BMI 1.021 0.937 1.113 0.6386 Note: ^([1])Cox Proportional Hazard ratio with 95% CI and p-value was calculated using PROC PHREG procedure.

Cox Proportional Hazards Regression Analysis for time to 100% wound closure in the PP population is summarised in Table 53 below. The effect of covariates estimated by the proportional hazards model is reported as hazard ratios. As per the hazards ratio and p-value, covariates such as treatment group showed an association with time to completion of wound closure (p-value=0.0077) and the subjects treated with Granexin™ Gel plus SoC would have wound closure 3.150 times more than the subjects treated with SoC only. Wound size, wound duration, baseline wound depth, and BMI were not associated with time to wound closure.

TABLE 53 Analysis of Time to First Complete (100%) Wound Closure (Cox's Proportional Hazards Regression Analysis) PP Population (N = 68) 95% CI for Hazard Ratio Statistics Hazard Ratio^([1]) Lower Upper p-value Treatment Group 3.150 1.354 7.326 0.0077 Wound size 0.999 0.997 1.000 0.0714 Wound duration 1.000 0.997 1.003 0.9942 Baseline wound depth 2.027 0.333 12.352 0.4435 BMI 1.021 0.935 1.114 0.6449 Note: ^([1])Cox Proportional Hazard ratio with 95% CI and p-value was calculated using PROC PHREG procedure.

Time to 50% wound closure in the ITT population is summarized in Table 54. As per the hazards ratio and p-value, covariates such as treatment group and wound size, showed an association with time to completion of wound closure (treatment group p-value=0.0143; wound size p-value=0.0162) and the subjects treated with Granexin™ Gel plus SoC would have wound closure 2.247 times more than the subjects treated with SoC only.

TABLE 54 Analysis of Time to First Complete (50%) Wound Closure (Cox's Proportional Hazards Regression Analysis) ITT Population (N = 92) 95% CI for Hazard Ratio Statistics Hazard Ratio^([1]) Lower Upper p-value Treatment Group 2.247 1.176 4.294 0.0143 Wound size 0.999 0.997 1.000 0.0162 Wound duration 1.000 0.997 1.002 0.7702 Baseline wound depth 0.680 0.182 2.537 0.5654 BMI 0.988 0.928 1.052 0.7046 Note: ^([1])Cox Proportional Hazard ratio with 95% confidence interval and p-value was calculated using PROC PHREG procedure.

Time to 50% wound closure in the mITT population is summarized in Table 55. As per the hazards ratio and p-value, covariates such as treatment group and wound size, showed an association with time to completion of wound closure (treatment group p-value=0.0067; wound size p-value=0.0346) and the subjects treated with Granexin™ Gel plus SoC would have wound closure 2.501 times more than the subjects treated with SoC only.

TABLE 55 Analysis of Time to First Complete (50%) Wound Closure (Cox's Proportional Hazards Regression Analysis) mITT Population (N = 77) 95% CI for Hazard Ratio Statistics Hazard Ratio^([1]) Lower Upper p-value Treatment Group 2.501 1.289 4.853 0.0067 Wound size 0.999 0.998 1.000 0.0346 Wound duration 1.000 0.997 1.002 0.8310 Baseline wound depth 0.805 0.217 2.991 0.7461 BMI 0.998 0.937 1.064 0.9585 Note: ^([1])Cox Proportional Hazard ratio with 95% confidence interval and p-value was calculated using PROC PHREG procedure.

Time to 50% wound closure in the PP population is summarized in Table 56. As per the hazards ratio and p-value, covariates such as treatment group and wound size, showed an association with time to completion of 50% wound closure (treatment group p-value=0.0059; wound size p-value=0.0442) and the subjects treated with Granexin™ Gel plus SoC would have wound closure 2.611 times more than the subjects treated with SoC only.

TABLE 56 Analysis of Time to First Complete (50%) Wound Closure (Cox's Proportional Hazards Regression Analysis) PP Population (N = 68) 95% CI for Hazard Ratio Statistics Hazard Ratio^([1]) Lower Upper p-value Treatment Group 2.611 1.319 5.169 0.0059 Wound size 0.999 0.998 1.000 0.0442 Wound duration 1.000 0.997 1.002 0.8788 Baseline wound depth 0.604 0.151 2.412 0.4755 BMI 0.998 0.935 1.064 0.9469 Note: ^([1])Cox Proportional Hazard ratio with 95% confidence interval and p-value was calculated using PROC PHREG procedure.

Wound Recurrence

Overall in ITT, mITT, and PP population, wound recurrence was reported in 10 subjects (Granexin™ Gel plus SoC group: 5 subjects; SoC group: 5 subjects) for the entire duration of the study. Of these 10 subjects, wound recurrence was observed in 2 subjects in Granexin™ Gel plus SoC group (Subject 504, Subject 907) and 2 subjects in SoC group (Subject 1003; Subject 1103) after completion of treatment at 12 weeks. There was no statistically significant difference in wound recurrence between the 2 treatment groups (p-value≧0.9999).

TABLE 57 Wound Recurrence in the Overall Population Treatment Group Granexin ™ Gel plus SoC SoC Overall Recurrence, n (%)^([1]) (N = 46) (N = 46) (N = 92) At any visit Yes  5 (10.9%)  5 (10.9%) 10 (10.9%) No 41 (89.1%) 41 (89.1%) 82 (89.1%) p-value^([2]) — ≧0.9999 — After Week 12 Yes 2 (4.3%) 2 (4.3%) 4 (4.3%) No 44 (95.7%) 44 (95.7%) 88 (95.7%) p-value^([2]) — ≧0.9999 — Note: ^([1])Percentage was calculated by taking respective column header group count as denominator. ^([2])p-value was calculated using the chi-square Test for comparison between two treatment groups.

Table 58 summarizes the time points of wound recurrence. Wound recurrence within 2 weeks was observed in 3 subjects (Subject 704, Subject 907, Subject 1002) in Granexin™ Gel plus SoC group and 2 subjects (Subject 208, Subject 902) in SoC group.

TABLE 58 Timepoints of Wound recurrence Visit Wound Subject Visit Wound Visit Wound Healed Number Treatment Group Healed recurred Again 206 Granexin ™ Gel plus Visit 9 Visit 13 — SOC 504 Granexin ™ Gel plus Visit 8 Visit 18 — SOC 704 Granexin ™ Gel plus Visit 10 Visit 11 — SOC 907 Granexin ™ Gel plus Visit 15 Visit 16 Visit 17 SOC 1002 Granexin ™ Gel plus Visit 7 Visit 8 Visit 12 SOC 208 SOC Visit 10 Visit 11 Visit 16 902 SOC Visit 8 Visit 9 Visit 10 1003 SOC Visit 9 Visit 16 — 1103 SOC Visit 10 Visit 16 Visit 17 1107 SOC Visit 11 Visit 13 —

The intensity of pain experienced by the subjects was recorded on a Visual Analogue Scale of 1 to 10 where 1 indicated “no pain” and 10 indicated “extreme pain” at all visits. The summary of subject self assessment of intensity of pain at all visits from Baseline to Week 12 for the ITT population is presented in and Table 59.

Table 59 summarizes the analysis of subject self assessment of intensity of pain at Week 12 for the ITT population. The intensity of pain was not statistically significantly higher in subjects treated with Granexin™ Gel plus SoC as compared to those treated with SoC only (p-value=0.8393).

TABLE 59 Analysis of Subject Self Assessment of Intensity of Pain at Week 12 ITT Population (N = 92) Treatment Group Granexin ™ Gel plus SoC group SoC group Statistics (N = 46) (N = 46) Intensity of pain at Week 12 n 39 38 Mean 0.46 0.34 SD 1.072 0.708 Median 0.00 0.00 Range (Min:Max) (0.0:5.0) (0.0:3.0) Missing 0 1 p-value^([1]) 0.8393 Note: ^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group.

Table 60 summarizes the analysis of subject self assessment of intensity of pain at Week 12 for the mITT population. The intensity of pain was not statistically significantly higher in subjects treated with Granexin™ Gel plus SoC as compared to those treated with SoC only (p-value=0.1663).

TABLE 60 Analysis of Subject Self Assessment of Intensity of Pain at Week 12 mITT Population (N = 77) Treatment Group Granexin ™ Gel plus SoC group SoC group Statistics (N = 41) (N = 36) Intensity of pain at Week 12 n 34 29 Mean 0.18 0.45 SD 0.387 0.783 Median 0.00 0.00 Range (Min:Max) (0.0:1.0) (0.0:3.0) Missing 0 1 p-value^([1]) 0.1663 Treatment Group Granexin ™ Gel plus SoC group SoC group Statistics (N = 46) (N = 46) Intensity of pain at Week 12 N 34 27 Mean 0.18 0.48 SD 0.387 0.802 Median 0.00 0.00 Range (Min:Max) (0.0:1.0) (0.0:3.0) Treatment Group Granexin ™ Gel plus SoC group SoC group Statistics (N = 41) (N = 36) Missing 0 1 p-value^([1]) 0.1195 Note: ^([1])p-value is evaluated by using non-parametric Wilcoxon Mann-Whitney U test between treatment group.

Wound Tracing were done for all subjects from Screening till Visit 15 or till the time the wound had healed. Qualitative Assessment of the wound site were done at the site at every visit for the presence of granulation, necrotic tissue, exudate levels, wound odor, and eschar as listed in the CRF.

The non-parametric Wilcoxon Mann-Whitney U test was used to analyze mean percent reduction of wound area from Baseline to Week 12 in both the treatment groups since the data did not follow a normal distribution, as confirmed by a Shapiro-Wilk p-value of <0.0001 and a non-linear Q-Q plot. The addition of ranks of both groups obtained after sorting the data of wound size reduction in ascending order showed that in Granexin™ Gel plus SoC subjects, the actual sum of scores was higher than the expected sum of scores (ITT: actual: 1210.00, expected: 1071.00; mITT: actual: 1136.00, expected: 990.00; PP: actual: 1123.00, expected: 973.50). In SoC group, the actual sum of scores was lower than the expected sum of the scores (ITT: actual: 743.00, expected: 882.00; mITT: actual: 634.00, expected: 780.00; PP: actual: 588.00; expected: 737.50). Therefore, subjects in Granexin™ Gel plus SoC group had a statistically significantly higher percent wound closure from Baseline to Week 12, as compared to subjects in SoC group (ITT p-value=0.0238; mITT p-value=0.0105; PP p-value=0.0073).

Non-parametric analysis for secondary endpoint showed that the mean percent reduction of wound area from Baseline to Week 4 in Granexin™ Gel plus SoC group was statistically significantly higher, as compared to subjects in SoC group (ITT p-value=0.0006; mITT p-value=0.0001; PP p-value<0.0001).

Time to complete (100%) wound closure by Week 12 was statistically significantly lesser in Granexin™ Gel plus SoC group, as compared to SoC group (ITT p-value=0.0006; mITT p-value<0.0001; PP p-value<0.0001). In Granexin™ Gel plus SoC group, 60.9% in the ITT population, 68.3% subjects in mITT population, and 80.0% subjects in the PP population had 100% wound closure by Week 12 as compared to 32.6% in the ITT population, 36.1% subjects in mITT population, and 36.4% in the PP population in the SoC group. The median duration of 100% wound closure was 6 weeks in ITT, mITT, and PP population in the Granexin™ Gel plus SoC group. In the SoC group, the median duration of time to complete wound closure was about 12.14 weeks in the ITT population and not achieved in the mITT and PP population.

The time taken to achieve 50% wound closure by Week 12 was statistically significantly lower in Granexin™ Gel plus SoC group as compared to SoC group subjects (ITT p-value=0.0002, mITT p-value<0.0001, PP p-value<0.0001). In Granexin™ Gel plus SoC group, 73.9% subjects in the ITT, 80.5% subjects in mITT population, and 91.4% subjects in the PP population had 50% wound closure and in SoC group, 54.3% subjects in ITT, 63.9% subjects in mITT population, and 66.7% in PP population had 50% wound closure. In ITT, mITT, and PP population, the median duration of 50% wound closure was 2.86 weeks in subjects treated with Granexin™ Gel plus SoC. Subjects treated with SoC alone had median duration of 6.86 weeks in the ITT population and 8 weeks in the both the mITT and PP population.

In Cox Proportional Hazard Regression Analysis for time to 100% wound closure covariates such as treatment group showed a significant association with time to completion of wound closure (ITT p-value=0.0406; mITT p-value=0.0139, PP p-value=0.0077) and the Granexin™ Gel plus SoC group subjects had ITT: 2.281; mITT p-value=2.776; PP: 3.150 times more chances of 100% wound closure than the SoC group subjects. Wound duration, wound size, baseline wound depth, and BMI were not significant factors affecting wound closure. Treatment group and wound size showed a significant association with time to completion of 50% wound closure (ITT: treatment group p-value=0.0143; wound size p-value=0.0162; mITT: treatment group p-value=0.0067; wound size p-value=0.0346; PP: treatment group p-value=0.0059; wound size p-value=0.0442) and the Granexin™ Gel plus SoC group subjects had ITT: 2.247; mITT: 2.501 times; PP: 2.61 times more chances of wound closure than the SoC group subjects. Wound duration, baseline wound depth, and BMI were not significant factors affecting 50% wound closure.

The categorical analysis of incidence of 100% wound closure at Week 12 was done using the Cochran-Mantel-Haenszel analysis with Breslow-Day test and the Chi-square test. The Chi-square test performed by combining the data from all the study centers showed that the number of responders with 100% wound closure in Granexin™ Gel plus SoC group (ITT: 56.5%; mITT: 63.4%; PP: 74.3%) was statistically significantly higher than in SoC group (ITT: 28.3%; mITT: 30.6%; PP: 30.3%) (ITT: p-value=0.0061; mITT p-value=0.0040; PP: p-value=0.0003).

The number of responders with 50% wound closure in Granexin™ Gel plus SoC group were 63% in ITT, 68.3% in mITT population as compared to 43.5% in ITT, 50% in mITT population in the SoC group which was not statistically significant (ITT p-value=0.0600; mITT p-value=0.1025). The number of responders with 50% wound closure in Granexin™ Gel plus SoC group in the PP population were 80% as compared to 51.5% in the SoC group which was statistically significantly higher (p-value=0.0131).

Hence, in the PP population, the frequency of 100% and 50% wound closure (epithelialization) was significantly higher in wounds treated with Granexin™ Gel plus SoC s than in SoC group subjects.

The intensity of pain experienced by the subjects was recorded on a Visual Analogue Scale of 1 to 10 where 1 indicated “no pain” and 10 indicated “extreme pain”. There was no statistically significant difference in subject self assessment of intensity of pain at the end of treatment at Week 12 between the 2 treatment groups (ITT p-value=0.8393; mITT p-value=0.1663, PP p-value=0.1195, Wilcoxon Mann-Whitney U test).

Brief Summary of Adverse Events

Overall, 28 subjects reported 40 AEs of which 24 AEs were reported by subjects in Granexin™ Gel plus SoC group; 16 AEs were reported in the SoC group. Out of 24 AEs, 21 (87.5%) in Granexin™ Gel plus SoC group and out of 16 AEs, 12 (75%) AEs in SoC group were mild. Three (12.5%) events in Granexin™ Gel plus SoC group and 3 (18.8%) events in SoC group were moderate; and 1 (6.3%) event in SoC group was severe. None of the AEs in either Granexin™ Gel plus SoC group or SoC group were related to the study drug or study treatment. Of the 40 events, 35 (87.5%) recovered and 1 (2.5%) event recovered with sequelae, 3 (7.5%) AEs were ongoing when the subject completed the study. No action was taken against 39 (97.5%) AEs. None of the subjects withdrew due to an AE and 1 (1.1%) subject in SoC group discontinued the study treatment due to death.

A total of 2 (2.2%) SAEs were reported in SoC group one of which was death due to MI and the other was Deep Vein Thrombosis. Both the SAEs were not related to the study treatment. No SAEs were reported in subjects in Granexin™ Gel plus SoC group (Table 61).

TABLE 61 Overview of Adverse Events-Safety Population (N = 92) Treatment Group Granexin ™ Gel plus SoC SoC Overall Statistics (N = 46) (N = 46) (N = 92) Total number of AEs 24 16 40 reported Subjects reporting any 14 (30.4%) 14 (30.4%) 28 (30.4%) AEs^([1]) Subjects reporting 1 AE  8 (17.4%) 12 (26.1%) 20 (21.7%) Subjects reporting >1 AE  6 (13.0%) 2 (4.3%) 8 (8.7%) Subjects Reporting No 32 (69.6%) 32 (69.6%) 64 (69.6%) AEs^([1]) Number of AEs with severity of:^([2]) Mild 21 (87.5%) 12 (75.0%) 33 (82.5%) Moderate  3 (12.5%)  3 (18.8%)  6 (15.0%) Severe 0 (0.0%) 1 (6.3%) 1 (2.5%) Number of AEs with relationship of:^([2]) Not Related  24 (100.0%)  16 (100.0%)  40 (100.0%) Unlikely 0 (0.0%) 0 (0.0%) 0 (0.0%) Possible 0 (0.0%) 0 (0.0%) 0 (0.0%) Probable 0 (0.0%) 0 (0.0%) 0 (0.0%) Definite 0 (0.0%) 0 (0.0%) 0 (0.0%) Number of AEs by outcome:^([2]) Recovered 22 (91.7%) 13 (81.3%) 35 (87.5%) Recovered with sequelae 0 (0.0%) 1 (6.3%) 1 (2.5%) On-going when subject 2 (8.3%) 1 (6.3%) 3 (7.5%) completed the study Death 0 (0.0%) 1 (6.3%) 1 (2.5%) Unknown 0 (0.0%) 0 (0.0%) 0 (0.0%) Number of AEs by action taken:^([2]) None  24 (100.0%) 15 (93.8%) 39 (97.5%) Discontinued study drug 0 (0.0%) 1 (6.3%) 1 (2.5%) Subjects reporting AEs 0 (0.0%) 0 (0.0%) 0 (0.0%) leading to withdrawal^([1]) Subjects reporting SAEs^([1]) 0 (0.0%) 2 (4.3%) 2 (2.2%) Subjects reporting death 0 (0.0%) 1 (2.2%) 1 (1.1%) Note: ^([1])Percentage was calculated by taking respective column header group count as denominator. ^([2])Percentage was calculated by taking count of ‘Total Number of AEs Reported’ in corresponding treatment group as denominator.

Display of Adverse Events

The AEs by MedDRA system organ class and preferred term are summarized in

There was no statistically significant difference in the number of subjects with at least 1 AE between the treatment groups (p-value≧0.9999). More than 5% safety population reported venous ulcer pain (10 [10.9%]) and wound complication (12 [13.0%] subjects).

TABLE 62 Summary of Adverse Events by MedDRA System Organ Class and Preferred Term Safety Population (N = 92) Treatment Group n (%)^([1]) Granexin ™ Gel plus System Organ Class/Preferred SoC SoC Overall Term (n = 46) (n = 46) (N = 92) p-value^([2]) Total number of subjects with at 14 (30.4%) 14 (30.4%) 28 (30.4%) ≧0.9999 least one AE Total number of AEs reported 24 16 40 Cardiac disorders 0 (0.0%) 1 (2.2%) 1 (1.1%) Myocardial infarction^([3]) 0 (0.0%)[0] 1 (2.2%)[1] 1 (1.1%)[1] General disorders and 0 (0.0%) 2 (4.3%) 2 (2.2%) administration site conditions Pyrexia 0 (0.0%)[0] 2 (4.3%)[2] 2 (2.2%)[2] Infections and infestations 2 (4.3%) 1 (2.2%) 3 (3.3%) Wound infection 2 (4.3%)[2] 1 (2.2%)[2] 3 (3.3%)[4] Injury, poisoning and procedural 2 (4.3%) 4 (8.7%) 6 (6.5%) complications Blister 1 (2.2%)[1] 0 (0.0%)[0] 1 (1.1%)[1] Wound complication 7 (15.2%)[11] 5 (10.9%)[5] 12 (13.0%)[16] Respiratory, thoracic and 2 (4.3%) 0 (0.0%) 2 (2.2%) mediastinal disorders Cough 1 (2.2%)[1] 0 (0.0%)[0] 1 (1.1%)[1] Pneumonitis 1 (2.2%)[1] 0 (0.0%)[0] 1 (1.1%)[1] Skin and subcutaneous tissue 7 (15.2%) 4 (8.7%) 11 (12.0%) disorders Venous ulcer pain 6 (13.0%)[6] 4 (8.7%)[4] 10 (10.9%)[10] Dermatitis allergic 1 (2.2%)[1] 0 (0.0%)[0] 1 (1.1%)[1] Pruritus 1 (2.2%)[1] 0 (0.0%)[0] 1 (1.1%)[1] Vascular disorders 0 (0.0%) 1 (2.2%) 1 (1.1%) Bleeding varicose vein 0 (0.0%)[0] 1 (2.2%)[1] 1 (1.1%)[1] Deep vein thrombosis 0 (0.0%)[0] 1 (2.2%)[1] 1 (1.1%)[1] Note: ^([1])Percentage was calculated by taking respective column header group count as denominator. ^([2])p-value was calculated by comparing two treatment group using Chi-square test. ^([3])Myocardial infarction led to death of the subject.

Of the 40 AEs reported in this study, one severe event of MI was reported by a subject in the SoC group None of the AEs (Granexin™ Gel plus SoC group: 24 [100%]; SoC group: 16 [100.0%] had any definite, probable, or possible relationship to Granexin™ Gel or the study treatment. One subject (Subject 1115) in SoC group was discontinued from the study treatment due to death. No other subjects were withdrawn or discontinued from the study due to an AE. Out of 40 AEs, 35 (87.5%) recovered, 1 (2.5%) AE (venous ulcer pain) in SoC group (Subject 102) recovered with sequelae, 3 (3.3%) AEs of wound complication (Granexin™ Gel plus SoC group: 2 subjects [Subject 702, 916]; SoC group: 1 subject [Subject 1113]) were on-going when the subjects completed the study, and 1 subject (Subject 1115, SoC group) died due to MI. One subject (Subject 1115, SoC group) died due to MI.

At Screening visit, of the 9 female subjects in the study, 2 were surgically sterilized and 4 were post menopausal. Pregnancy test was done for 3 (3.3%) subjects and doppler waveform analysis for all 92 subjects. The mean Doppler waveform reading for Granexin™ Gel plus SoC group subjects was 11.60 cm/s and 9.57 cm/s for SoC group subjects. X-ray was taken for 86 (93.5%) subjects where X-ray finding was normal for 81 (88.0%) and abnormal for 5 (5.4%) subjects, which was not clinically significant. None of the target wounds were infected with βhemolytic Streptococcus culture. The eGFR for males in Granexin™ Gel plus SoC group was 95.18 mL/min and 88.50 mL/min in SoC group subjects. However, the eGFR for female and ABPI were comparable between the treatment groups.

None of the subjects in the study had clinically significant hematological parameters at Screening visit or Week 12. Two subjects in Granexin™ Gel plus SoC group had clinically significant HbA1c at Screening visit but none of the subjects had clinically significant HbA1c at 12 weeks. None of the subjects had any clinically significant creatinine, uric acid, BUN, potassium, sodium, chloride, bicarbonate, albumin, AST, ALT, and total cholesterol at Screening visit or at Week 12.

Immunogenicity Testing: Using universal precautions recommended for biological samples, the blood samples were collected in yellow top gel vaccutainers. They were properly labeled with date and time of collection, site number, visit number, screening and randomization number, and the sample type. Using acceptable venipuncture technique 7 mL of whole blood was collected and allowed to stand for 15 min for the clot to form. Serum was separated by centrifuging the blood samples at 3500 rpm. Approximately 2 mL of the separated serum was pipetted out into two aliquots and refrigerated at 2-6° C. till shipped. These tubes were labeled with date and time of collection, site number, visit number, screening and randomization number, and the sample type. These samples were shipped to a central storage facility (Metropolis Laboratory, Mumbai) under appropriate temperature conditions with dry ice or frozen cold packs.

The samples were stored at −20° C. at Metropolis and shipped in batches to WuXi Laboratories at Philadelphia, Pa., under appropriate temperature conditions with serum sample shipment forms.

Anti-ACT1 antibodies were not detected in the serum in any of the subjects at Screening and Week 12.

The systolic and diastolic blood pressure, heart rate, respiratory rate, and body temperature were comparable between the treatment groups at Screening visit and at Week 12.

In the overall safety population, the ECG was normal in 81 (88.0%) subjects. Changes in ECG impression reported in the remaining subjects were not clinically significant at the Screening visit (End-of-Text Table 14.3.6)

Overall, 28 (30.4%) subjects reported 40 AEs of which 24 AEs were reported by subjects in Granexin™ Gel plus SoC group; 16 AEs were reported in the SoC group. Out of 24 AEs, 21 (87.5%) in Granexin™ Gel plus SoC group and out of 16 AEs, 12 (75%) AEs in SoC group were mild. Three (12.5%) events in Granexin™ Gel plus SoC group and 3 (18.8%) events in SoC group were moderate; and 1 (6.3%) event in SoC group was severe. None of the AEs in either Granexin™ Gel plus SoC group or SoC group were related to the study drug or study treatment. Of the 40 events, 35 (87.5%) recovered and 1 (2.5%) event recovered with sequelae. None of the subjects withdrew due to an AE and 1 (1.1%) subject in SoC group discontinued the study treatment due to death.

A total of 2 (2.2%) SAEs were reported in SoC group one of which was death due to Myocardial Infarction and the other was Deep Vein Thrombosis. Both the SAEs were not related to the study treatment. No SAEs were reported by subjects in Granexin™ Gel plus SoC group Overall, there was no significant difference in the number of subjects reporting AEs between the 2 treatment groups (Granexin™ Gel plus SoC group, 14; SoC group, 14; p-value≧0.9999).

At Screening visit, no clinically significant abnormalities were observed in any of the treatment groups. The vital signs and ECG were normal and comparable between both the treatment groups. Anti-ACT1 peptide antibodies were not detected in any of the subjects in both Screening and EOS serum samples.

This was a double blind, randomized, prospective, parallel group, multi-center Phase II study conducted at 10 centers in India to evaluate the efficacy and safety of Granexin™ Gel in the treatment of VLU. The total duration of the study was 11 months, including 5 months enrollment period and 6 months for study procedures. The study duration for individual subjects was 6 months which included 18 visits. A total of 101 subjects with VLU were screened for this study, 5 were found to be screen failures, and 4 subjects did not initiate participation in the study. A total of 92 subjects enrolled in the study had mean wound duration of 68.4 weeks at Screening and mean wound area of 353.64 mm² at Baseline. These subjects were randomized to receive Granexin™ Gel plus SoC or SoC alone for the treatment of VLU. Of these, 70 completed the study and 22 subjects did not complete the study due to consent withdrawal, loss to follow-up, non-compliance, or SAE.

Of 92 enrolled subjects, 15 subjects met exclusion criterion number 1 or did not have adequate evaluable wound photographic data post randomization but were considered in the Safety and ITT population. These subjects were excluded from the mITT and PP populations. A total of 83 (90.2%) subjects were males and 9 (9.8%) were females. The mean age of the study population was 49.8 years, mean weight was 70.47 kg, mean height was 168.35 cms, and mean BMI was 24.79 kg/m².

The primary efficacy endpoint of the study was to evaluate mean percent wound closure from Baseline (Visit 2) to Week 12 (Visit 15). The non-parametric Wilcoxon Mann-Whitney U test was used to analyze mean percent reduction of wound area from Baseline to Week 12 in both the treatment groups since the data did not follow a normal distribution, as confirmed by a Shapiro-Wilk p-value<0.0001 and a non-linear Q-Q plot. Subjects treated with Granexin™ Gel plus SoC showed statistically significantly higher percent wound closure from Baseline to Week 12 compared to subjects treated with SoC alone (ITT p-value=0.0238; mITT p-value=0.0105; PP p-value=0.0073), suggesting that at Week 12, wounds treated with Granexin™ Gel plus SoC had a significant reduction in size as compared to wounds treated with SoC alone.

Mean percent wound closure at Week 4, time to complete (100% and 50%) wound closure, incidence of complete wound closure, and subject self assessment of pain were also analyzed. At Week 4, the wound area percent of reduction in Granexin™ Gel plus SoC group was statistically significantly higher than in SoC group (ITT p-value=0.0006; mITT p-value=0.0001; PP p-value<0.0001 Wilcoxon Mann-Whitney U test).

Subjects treated with Granexin™ Gel plus SoC achieved 100% wound closure in median duration of 6 weeks in ITT, mITT, and PP population which was significantly faster compared to the SoC group in which the median duration was 12.14 weeks in the ITT population and not achieved in both mITT and PP population during the 12 weeks of efficacy assessments (ITT p-value=0.0006; mITT p-value=0.0001; PP p-value<0.0001; log-rank test).

Subjects treated with Granexin™ Gel plus SoC achieved 50% wound closure in 2.86 weeks in both ITT and PP population while subjects treated with SoC alone achieved it in 6.86 weeks in ITT population and 8 weeks in both mITT and PP population, respectively (ITT p-value=0.0002, mITT p-value<0.0001, PP p-value<0.0001). The categorical analysis of incidence of 100% and 50% wound closure at Week 12 was conducted and the subjects who had 100% wound closure were considered as responders. Overall in the ITT, mITT, and PP populations, 56.5%, 63.4%, and 74.3% of the subjects in Granexin™ Gel plus SoC group, respectively, responded to treatment with 100% wound closure as compared to 30.3% of the subjects in the SoC group. The number of responders were significantly higher in Granexin™ Gel plus SoC group than in the SoC group (ITT: p-value=0.0061; mITT p-value=0.0040; PP: p-value=0.0003; Chi-square analysis). Similarly, in the PP population, 80.0% of the subjects in Granexin™ Gel plus SoC group responded to treatment and achieved 50% wound closure as compared to 51.5% of the subjects in the SoC group. The number of responders with 50% wound closure were significantly higher in Granexin™ Gel plus SoC group than in the SoC group (p-value=0.0131; Chi-square analysis), indicating that the wounds treated with Granexin™ Gel plus SoC have higher incidences of 100% and 50% wound closure (epithelialization) than wounds treated with SoC alone.

Cox Proportional Hazard Regression Model analysis was conducted to evaluate if one or more covariates such as treatment group, wound duration, baseline wound depth, and BMI etc. were associated with the time to complete 100% or 50% wound closure. It showed that treatment group was a significant factor affecting wound closure and Granexin™ Gel plus SoC group subjects had ITT: 2.281; mITT: 2.776; PP: 3.150 times more chances of 100% wound closure and ITT: 2.247; mITT: 2.501; PP: 2.61 times more chances of 50% wound closure than the SoC group subjects.

At every visit, self assessment of intensity of pain score was completed by each subject and the scores were given in comparison to the last visit. Therefore significant change in the pain scores was not observed and there was no statistically significant difference in intensity of pain as assessed by the subject at Week 12 between the 2 treatment groups.

The findings from the present study demonstrated a good safety profile with Granexin™ Gel. Overall, 28 subjects reported 40 AEs of which 24 AEs were reported by subjects in Granexin™ Gel plus SoC group; 16 AEs were reported in the SOC group. The only 2 SAEs reported, were in the SoC group. One SAE was Myocardial infarction which resulted in death, and the other SAE was Deep vein thrombosis. Of the 40 AEs, 35 (87.5%) recovered. None of the AEs were related to Granexin™ Gel. None of the subjects were withdrawn from the study due to an AE.

No clinically significant abnormalities in the laboratory parameters were observed in either of the treatment groups. The vital signs and ECG were normal and comparable between both the treatment groups. Anti-ACT1 peptide antibodies were not detected in the serum samples collected at Screening or at Week 12.

In conclusion, Granexin™ Gel along with SoC has shown to accelerate wound healing in subjects with VLU, making it an efficacious, safe, and well tolerated therapeutic option in the treatment of VLU.

Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications cited herein are incorporated herein by reference for the purpose of disclosing and describing specific aspects of the invention for which the publication is cited.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

TABLE 9 Wound Healing Gel Qualitative and Quantitative Composition Concentration Ingredients Grade Function (% w/w) Grams/kg Batch Peptide 328967 (ACT1) — Active 0.0072; 0.018 0.072; 0.18 0.036; 0.072 0.36; 0.72 Methylparaben NF Preservative 0.17 1.7 Propylparaben NF Preservative 0.02 0.2 Glycerin USP Solvent 5 50 Sodium Phosphate Monobasic USP Buffer Agent 0.263 2.63 Sodium Phosphate Dibasic USP Buffer Agent 0.044 0.44 Propylene Glycol USP Solvent 3 30 Edetate Disodium (EDTA) USP Chelating Agent 0.05 0.5 D-Mannitol USP Stabilizer 0.05 0.5 Hydroxyethylcellulose, 250HHX NF Gelling Agent 1.25 12.5 Purified Water, qsad USP Solvent 1 1 kg 

The invention claimed is:
 1. A method of treating a chronic wound in a subject, comprising administering to the subject a topical formulation comprising at least one alpha connexin polypeptide, wherein the at least one polypeptide comprises the carboxy terminal-most 4 to 30 contiguous amino acids of an alpha Connexin, or a conservative variant comprising up to 2 conservative substitutions, and wherein the formulation further comprises hydroxyethylcellulose gel.
 2. The method of claim 1, wherein the formulation is administered in a dosing regimen at day 0, day 3, week 1, week 2, week 3, week 4, week 5, week 6, week 7, week 8, week 9, week 10, week 11, and week
 12. 3. The method of claim 1, wherein the hydroxyethylcellulose gel is present at a concentration of about 1.25% (w/w).
 4. The method of claim 1, wherein the alpha connexin polypeptide is connexin 37, connexin 40, connexin 43, or connexin
 45. 5. The method of claim 1, wherein the alpha connexin polypeptide is linked at its amino terminus to a cellular internalization transporter.
 6. The method of claim 5, wherein the cellular internalization transporter is an antennapedia sequence.
 7. The method of claim 1, wherein the formulation comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:
 9. 8. The method of claim 1, wherein the chronic wound is an ulcer.
 9. The method of claim 1, wherein the chronic wound is a lower extremity ulcer.
 10. The method of claim 1, wherein the chronic wound is selected from the group consisting of venous leg ulcers, diabetic foot ulcers, and pressure ulcers.
 11. The method of claim 1, wherein the method reduces the symptoms of the chronic wound.
 12. The method of claim 1, wherein the formulation does not induce excessive levels of side effects.
 13. The method of claim 1, wherein the method reduces the time to 100% wound closure as compared to standard of care treatment for wound healing.
 14. The method of claim 1, wherein the method reduces the time to 50% wound closure at compared to standard of care treatment for wound healing.
 15. The method of claim 1, wherein the method increases the average percent of wound closure at 12 weeks, as compared to standard of care treatment for wound healing.
 16. The method of claim 1, wherein the method reduces wound area as compared to the wound area reduced by standard of care treatment.
 17. The method of claim 1, wherein the chronic wound is treated in the absence of clinically significant abnormalities.
 18. The method of claim 1, wherein the formulation does not induce production of anti-alpha connexin polypeptide antibodies in the subject.
 19. The method of claim 1, wherein the method increases the incidence or frequency of 100% complete wound closure compared to standard of care treatments for wound healing.
 20. The method of claim 1, wherein the method is used to treat an ulcer lacking sufficient wound size reduction within one, four, 12, or 24 weeks of standard of care.
 21. The method of claim 1, wherein the method is used to treat an ulcer with less than 50% wound closure within one four, 12 or 24 weeks of standard of care.
 22. The method of claim 1, wherein the method is used to treat a patient with an ulcer possessing wound area and duration characteristics of a chronic wound.
 23. The method of claim 1, wherein the method is used to treat a patient with an ulcer possessing a wound with a non-healing wound trajectory.
 24. The method of claim 1, wherein the method is used to treat an ulcer possessing wound area and time duration characteristics of a chronic wound.
 25. The method of claim 1, wherein the formulation is administered daily or weekly.
 26. The method of claim 1, wherein the formulation is administered in a dosing regimen at day 0, day 3, day 7, and weekly thereafter until healing of the chronic wound is apparent. 